Compositions and methods involving methyladenosine phosphorylase in the diagnosis and treatment of proliferative disorders

ABSTRACT

Disclosed are novel nucleic acid and peptide compositions comprising methylthioadenosine phosphorylase (MTAP) and methods of use for MTAP amino acid sequences and DNA segments comprising MTAP in the diagnosis of human cancers and development of MTAP-specific antibodies. Also disclosed are methods for the diagnosis and treatment of tumors and other proliferative cell disorders, and identification of tumor suppressor genes and gene products from the human 9p21-p22 chromosome region. Such methods are useful in the diagnosis of multiple tumor types such as bladder cancer, lung cancer, breast cancer, pancreatic cancer, brain tumors, lymphomas, gliomas, melanomas, and leukemias.

The present application is a continuation of U.S. patent applicationSer. No. 08/674,311, filed Jul. 1, 1996, now issued as U.S. Pat. No.6,870,037, which is a continuation-in-part of U.S. Provisional PatentApplication 60/000,831, filed Jul. 3, 1995, the entire contents of eachof which are incorporated herein by reference in their entirety.

The United States government has certain rights in the present inventionpursuant to Grant DE-FG02-86ER60408 from the Department of Energy, GrantCA14599-19 from the National Cancer Institute, and Grants CA40046 andCA42557 from the United States Public Health Service.

1. BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to the field of molecularbiology. More specifically, it concerns novel amino acid sequencescomprising the human methylthioadenosine phosphorylase (MTAP) enzyme,and the nucleic acid segments comprising the MTAP gene. Disclosed aremethods and compositions related to cancer therapy involving the tumorsuppressor region of human chromosome 9p21.

1.2 Description of the Related Art

1.2.1 Tumors from 9p Abnormalities

Unbalanced translocations or interstitial deletions of 9p are recurringabnormalities in a variety of tumor types including acute lymphoblasticleukemia, glioma, melanoma, non-small cell lung cancer, head and neckcancer, bladder cancer and mesothelioma (Mitelman, 1994). Homozygousdeletions of DNA sequences on 9p or loss of heterozygosity have now beendescribed in multiple tumor types (Díaz et al., 1988; 1990; Olopade etal., 1992; 1993; Kamb et al., 1994; Nobori et al., 1994). A number ofthe cell lines and patient samples with 9p gene deletions also lack MTAPenzyme activity. The gene encoding MTAP had not been cloned but waspreviously mapped to 9p22-9q13 (Carerra et al., 1984). In a few cases,the deletions that included both the IFN gene cluster and the MTAP genewere interstitial and submicroscopic, suggesting that these genes or atumor suppressor gene closely linked to them were the target of the 9pdeletions. This hypothesis was further supported by the linkage of agene that confers susceptibility to melanoma (MLM) to 9p21 in the regionbetween D9S126 and the IFNA gene cluster (Cannon-Albright et al., 1992).

CDKN2 (p16^(INK4A)) was recently proposed as a candidate TSG in thislocus because the gene has been shown to be rearranged, deleted ormutated in a majority of tumor cell lines (Kamb et al., 1994; Nobori etal., 1994). This gene codes for a 16 kDa protein (p16) that inhibitsCDK4 and CDK6 by binding in competition with cyclin D (Serrano et al.,1993). In humans, CDKN2 is adjacent to a gene encoding a similarprotein, now called CDKN2B (p16^(INK4B)) which shares 44% homology withCDKN2 in the first 50 amino acids and up to 97 percent homology in theremainder of the protein (Hannon and Beach, 1994). Whether CDKN2 is MLMremains unclear, because two recent studies provide conflictingevidence. Hussussian et al. described six different disease-relatedgermline mutations in CDKN2 in 33 of 36 melanoma cases from 9 familiesand suggested that CDKN2 likely is MLM (Hussussian et al., 1994). Thisis in contrast to 2/13 mutations in 9p21-linked families and 0/38familial melanoma cases described (Kamb et al., 1994). These reportsraise the possibility that CDKN2 may not be the only clinically relevantTSG on 9p and that loss of tumor suppression may involve inactivation ofother as yet unidentified genes in the region in certain tumor types.Two additional regions of non overlapping homozygous deletions on 9p21were found in malignant mesothelioma; one telomeric to CDKN2 and theother centromeric to it (Cheng et al., 1994).

The coincident loss of MTAP enzyme activity in many tumor cell lineswith homozygous IFN gene deletions suggests that MTAP is closely linkedto the IFN gene cluster. It was suggested that 9p TSG should belocalized between the IFN gene cluster and the MTAP locus based on IFNgene rearrangements seen in two cell lines and leukemia cells from onepatient with deletions on 9p (Olopade et al., 1992). In the reportspublished to date, it has been difficult to determine the exact positionof the MTAP gene in relation to the homozygous deletions on 9p.

1.2.2 Transformation of Tumor Cells

The malignant transformation of tumor cells is known to be driven by theaccumulation of various genetic alterations including numerical andstructural chromosomal alterations. Among the specific alterationsassociated with neoplasms, the loss of tumor suppressor genes has beenrecognized as an important component. However, at present, deletions ormutations of the two commonly inactivated tumor suppressor genes, TP53and RB¹, have been detected only in the minority of patients with acutelymphoblastic leukemia (ALL) (Ahuja et al., 1991; Fenaux et al., 1992).

In contrast, cytogenetic deletions of chromosomal band 9p21 have beendetected in 10–15% of ALL cases indicating the presence of a novel tumorsuppressor gene in this region (Kowalczyk, 1983; Chilcote, 1985; Pollak,1987). The IFN gene cluster which is located on 9p21 was found to bedeleted in 43% of leukemia derived cell lines and 29% of primaryleukemia samples (Díaz et al., 1988; 1990). Recently, the CDKN2 gene(p16^(INK4A), MTS I, CDK4I) which encodes an inhibitor of thecyclin-dependent kinase 4, has been found to be homozygously deleted intumor cell lines and has been proposed as a candidate tumor suppressorgene in this region (Serrano et al., 1993; Kamb et al., 1994; Nobori etal., 1994). However, the frequency of point mutations of this gene inhematological malignancies has been very low suggesting that CDKN2 maybe not the critically relevant gene on 9p (Quesnel et al., 1994).Moreover, the extent of the homozygous deletions on 9p have not beenclearly delineated in primary tumors. 20 hematological malignancies wereexamined with cytogenetically well characterized 9p rearrangements todefine the critical region of 9p.

1.2.3 Deletions in 9p21

There are now numerous reports of large homozygous or hemizygousdeletions involving 9p21 in diverse types of tumors. The shortest regionof overlap in the different tumor types varies and covers a large regionon 9p21 usually from D9S171 to D9S162. Cannon-Albright et al. haverecently refined their location for the melanoma susceptibility gene(MLM) to a region between D9S736 and D9S171, a genetic distance of atleast 2 cM. Ishiiki et al. have also defined an interstitial deletionbetween D9S162 and D9S169, a region of 21 cM in the tumor from a patientwith familial melanoma This region encompassed both the regionhomozygously deleted in melanoma cell lines and the MLM locus, and alsooverlapped the hemizygous germline deletion seen in lymphocytes from thepatient described by Petty et al. (1993) with eight primary melanomas.In bladder cancer, a putative tumor suppressor gene locus involved inbladder tumorigenesis was localized to a 10 cM region flanked by D9S162and D9S171 (Cairns et al., 1994). Some investigators contend that thecritical region on 9p is proximal to D9S171 while others believe theregion to be telomeric to the IFN gene cluster.

The high frequency of homozygous or hemizygous deletions of this regionof 9p in human tumors suggests an advantage to inactivate this locus inmalignant cells. Each of the three genes identified thus far in thisregion appears to have some biological role in cancer. Therefore, theirinclusion in the deleted region may be advantageous to the malignantcell. Alternatively, intrinsic fragility or recombinogenicity aroundthis tumor suppressor locus may make the region a hot spot forillegitimate recombination during cell division. A fragile site has beenmapped to this region of 9p. Under certain culture conditions, breaksand gaps occur at a high frequency at fragile sites because they arehighly recombinogenic. Although the biological consequences of fragilesite expression are not fully understood, several lines of evidencepoint to their involvement in carcinogenesis. Therefore, it is possiblethat these deletions are related to the previously mapped fragile siteat 9p21.

It has been suggested that the preferred mechanism for gene inactivationin this locus is homozygous deletion rather than point mutations. Thatthere may be more than one tumor suppressor gene in this regionaccounting for this phenomenon is possible. If the inactivation of morethan one tumor suppressor gene is required to give the cells a growthadvantage, then deletions will be favored over point mutations. If twotumor suppressor genes are closely linked in a particular chromosomalregion, a deletion will frequently inactivate both at the same time,while point mutations can only inactivate one at a time, and willrequire two of these mutations to inactivate both genes. If the geneshave a low mutation rate e.g., 10⁻⁸, then the likelihood of mutatingboth genes is 10⁻⁸×10⁻⁸=10⁻¹⁶ which will be a rare event.

1.3 Deficiencies in the Prior Art

The problem of searching for additional genes on 9p21 in the face of asstrong a candidate as CDKN2 seems daunting. However, the fact remainsthat there are still several unanswered questions regarding its role intumor suppression. The mechanisms and genes involved in 9p deletions arecomplex and may not conform to the usual way of analyzing this problem.The characterization of tumor suppressor loci on 9p with regard to thegenes included in the deletions would represent a major advancement inthis area of tumor biology and cancer therapeutics. Determination of themechanism(s) responsible for the propensity of this genomic region toundergo frequent deletions is critical in the development of tumorsuppression therapies involving the 9p region. Likewise, DNA sequenceanalysis and elucidation of the amino acid sequence of MTAP wouldrepresent a major breakthrough in the development of novel genetherapies and medical diagnostics in the area of tumor suppression andcell proliferation disorder treatments.

2. SUMMARY OF THE INVENTION

The present invention overcomes one or more of these and other drawbacksinherent in the prior art by providing methods and compositions oftumor-associated genes and encoded gene products useful in tumordiagnosis. A defined region of 9p21 has been identified in which arelocated several putative tumor suppressor genes. Additionally, theentire MTAP gene has been sequenced thereby allowing development oftumor diagnostics and therapies designed to regulate suchtumor-associated genes.

In certain aspects the invention includes methods of identifying varioustypes of tumors. For example, tumors may be differentiated by comparingDNA patterns obtained from restriction analysis with several selectedrare endonucleases. Thus patterns from normal tissue DNA and tumortissue will differ as will DNA patterns from different types of tumorsin a generally reproducible manner for defined conditions with theselected endonucleases.

In other aspects of the invention, methods of gene therapy arecontemplated; particularly in the use of MTAP gene or fragments of MTAPgene or other tumor suppressor genes in the 9p21 region to inhibitexpression of oncogenes.

Additionally, the polypeptide products of the MTAP gene and other tumorsuppressor genes may be utilized to detect the presence of tumors.Assays may be conducted with antibody assays employing polyclonal ormonoclonal antibodies.

It is hypothesized that the cytogenetically visible abnormalities of 9pon one chromosomal allele might unmask a mutant recessive allele of thetumor suppressor gene on the cytogenetically normal homologue. Southernblot analysis was used to determine the frequency and size of these 9pdeletions using four markers generated from a 2.8 megabase YAC contig on9p21. The results were confirmed by interphase FISH analysis. The caseswith loss of one allele were analyzed by SSCP to detect point mutationsof the remaining CDKN2 allele.

The invention may be employed to promote expression of a desired gene inbone cells or tissues and to impart a particular desired phenotype tothe cells. This expression could be increased expression of a gene thatis normally expressed (i.e., “over-expression”), or it could be used toexpress a gene that is not normally associated with a particular cell inits natural environment. Alternatively, the invention may be used tosuppress the expression of a gene that is naturally expressed in suchcells and tissues, and again, to change or alter the phenotype. Genesuppression may be a way of expressing a gene that encodes a proteinthat exerts a down-regulatory function, or it may utilize antisensetechnology.

The invention provides reliable diagnostic methods and kits to test forthe presence of MTAP protein in vitro and in vivo. MTAP may serve as amarker for inactivation of the 9p21 locus in primary tumors.

Analysis of MTAP protein expression in human tumor cell lines usinganti-MTAP antisera raised against peptides in the NH₂-terminal region ofthe protein has identified novel reagents which are useful in exploringselective chemotherapy in tumors with high incidence of MTAP deficiencysuch as pancreatic cancer, melanomas, lymphomas, leukemias, gliomas, aswell as provide diagnostic tools for the identification of bladder,brain, breast, and lung cancers.

2.1 Tumor Suppressor Genes

As used herein, the term “tumor suppressor genes” is used to refer to agene or DNA coding region that encodes a protein, polypeptide or peptidethat is capable of suppressing, or assisting in the suppression of,tumor formation, or one that increases the rate of tumor suppression.

In general terms, a tumor suppressor gene may also be characterized as agene capable of suppressing the growth rate, proliferation, orregeneration rate of a cancerous cell such as, e.g., a melanoma, tumor,glioma, carcinoma, and the like. Thus, in certain embodiments, themethods and compositions of the invention may be employed to suppressthe growth or proliferation of a cancerous tissue or the cells of whichit is composed.

To prepare a tumor suppressor gene segment or cDNA one may follow theteachings disclosed herein and also the teachings of any of patents orscientific documents specifically referenced herein. One may obtain aDNA segment using molecular biological techniques, such as polymerasechain reaction (PCR™) or screening a cDNA or genomic library, usingprimers or probes with sequences based on the corresponding nucleotidesequence. The practice of such techniques is a routine matter for thoseof skill in the art, as taught in various scientific articles, such asSambrook et al. (1989), incorporated herein by reference. Certaindocuments further particularly describe suitable mammalian expressionvectors, e.g., U.S. Pat. No. 5,168,050, incorporated herein byreference.

It is also contemplated that one may clone further genes or cDNAs thatencode a tumor suppressor protein or polypeptide. The techniques forcloning DNA molecules, i.e., obtaining a specific coding sequence from aDNA library that is distinct from other portions of DNA, are well knownin the art. This can be achieved by, for example, screening anappropriate DNA library. The screening procedure may be based on thehybridization of oligonucleotide probes, designed from a considerationof portions of the amino acid sequence of known DNA sequences encodingrelated tumor suppressor proteins. The operation of such screeningprotocols are well known to those of skill in the art and are describedin detail in the scientific literature, for example, in Sambrook et al.(1989), incorporated herein by reference.

Tumor suppressor genes with sequences that vary from those described inthe literature are also encompassed by the invention, so long as thealtered or modified gene still encodes a protein that functions tosuppress tumor cells in any direct or indirect manner. These sequencesinclude those caused by point mutations, those due to the degeneraciesof the genetic code or naturally occurring allelic variants, and furthermodifications that have been introduced by genetic engineering, i.e., bythe hand of man.

Techniques for introducing changes in nucleotide sequences that aredesigned to alter the functional properties of the encoded proteins orpolypeptides are well known in the art, e.g., U.S. Pat. No. 4,518,584,incorporated herein by reference, which techniques are also described infurther detail herein. Such modifications include the deletion,insertion or substitution of bases, and thus, changes in the amino acidsequence. Changes may be made to increase the suppression activity of aprotein, to increase its biological stability or half-life, to changeits glycosylation pattern, and the like. All such modifications to thenucleotide sequences are encompassed by this invention.

It will, of course, be understood that one or more than one tumorsuppressor gene may be used in the methods and compositions of theinvention. The nucleic acid delivery methods may thus entail theadministration of one, two, three, or more such genes. The maximumnumber of genes that may be applied is limited only by practicalconsiderations, such as the effort involved in simultaneously preparinga large number of gene constructs or even the possibility of eliciting asignificant adverse cytotoxic effect. For example, particularcombinations of genes may be used to produce the most desirable effects,e.g., the combination of two or more distinct tumor suppressor genes.

In using multiple genes, they may be combined on a single geneticconstruct under control of one or more promoters, or they may beprepared as separate constructs of the same of different types. Thus, analmost endless combination of different genes and genetic constructs maybe employed. Certain gene combinations may be designed to, or their usemay otherwise result in, achieving synergistic effects on tumor cellsuppression, any and all such combinations are intended to fall withinthe scope of the present invention. Indeed, many synergistic effectshave been described in the scientific literature, so that one ofordinary skill in the art would readily be able to identify likelysynergistic gene combinations, or even gene-protein combinations.

It will also be understood that, if desired, the nucleic acid segment orgene could be administered in combination with further agents, such as,e.g., proteins or polypeptides or various pharmaceutically activeagents. So long as genetic material forms part of the composition, thereis virtually no limit to other components which may also be included,given that the additional agents do not cause a significant adverseeffect upon contact with the target cells or tissues. The nucleic acidsmay thus be delivered along with various other agents as necessary.

2.2 MTAP-Encoding DNA Segments

The present invention, in a general and overall sense, concerns theisolation and characterization of a novel gene, MTAP which encodes themethylthioadenosine phosphorylase, MTAP. A preferred embodiment of thepresent invention is a purified nucleic acid segment that encodes aprotein having an amino acid sequence as shown in FIG. 3B, and inaccordance with SEQ ID NO:2. Another embodiment of the present inventionis a purified nucleic acid segment, further defined as including anucleotide sequence in accordance with SEQ ID NO:1.

In a more preferred embodiment the purified nucleic acid segmentconsists essentially of the nucleotide sequence of SEQ ID NO:1. As usedherein, the term “nucleic acid segment” and “DNA segment” are usedinterchangeably and refer to a DNA molecule which has been isolated freeof total genomic DNA of a particular species. Therefore, a “purified”DNA or nucleic acid segment as used herein, refers to a DNA segmentwhich contains a MTAP coding sequence yet is isolated away from, orpurified free from, total genomic DNA, for example, total cDNA or humangenomic DNA. Included within the term “DNA segment”, are DNA segmentsand smaller fragments of such segments, and also recombinant vectors,including, for example, plasmids, cosmids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified MTAP generefers to a DNA segment including MTAP coding sequences isolatedsubstantially away from other naturally occurring genes or proteinencoding sequences. In this respect, the term “gene” is used forsimplicity to refer to a functional protein, polypeptide or peptideencoding unit. As will be understood by those in the art, thisfunctional term includes both genomic sequences, cDNA sequences orcombinations thereof. “Isolated substantially away from other codingsequences” means that the gene of interest, in this case MTAP, forms thesignificant part of the coding region of the DNA segment, and that theDNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or cDNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regionslater added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences which encode a MTAPgene, that includes within its amino acid sequence an amino acidsequence in accordance with SEQ ID NO:2. Moreover, in other particularembodiments, the invention concerns isolated DNA segments andrecombinant vectors incorporating DNA sequences which encode a gene thatincludes within its amino acid sequence the amino acid sequence of aMTAP gene corresponding to human MTAP.

Another preferred embodiment of the present invention is a purifiednucleic acid segment that encodes a protein in accordance with SEQ IDNO:2, further defined as a recombinant vector. As used herein the term,“recombinant vector”, refers to a vector that has been modified tocontain a nucleic acid segment that encodes a MTAP protein, or afragment thereof. The recombinant vector may be further defined as anexpression vector comprising a promoter operatively linked to theMTAP-encoding nucleic acid segment.

A further preferred embodiment of the present invention is a host cell,made recombinant with a recombinant vector comprising a MTAP gene. Therecombinant host cell may be a prokaryotic cell. In a more preferredembodiment, the recombinant host cell is a eukaryotic cell. As usedherein, the term “engineered” or “recombinant” cell is intended to referto a cell into which a recombinant gene, such as a gene encoding MTAP,has been introduced. Therefore, engineered cells are distinguishablefrom naturally occurring cells which do not contain a recombinantlyintroduced gene. Engineered cells are thus cells having a gene or genesintroduced through the hand of man. Recombinantly introduced genes willeither be in the form of a cDNA gene (i.e., they will not containintrons), a copy of a genomic gene, or will include genes positionedadjacent to a promoter not naturally associated with the particularintroduced gene.

Generally speaking, it may be more convenient to employ as therecombinant gene a cDNA version of the gene. It is believed that the useof a cDNA version will provide advantages in that the size of the genewill generally be much smaller and more readily employed to transfectthe targeted cell than will a genomic gene, which will typically be upto an order of magnitude larger than the cDNA gene. However, one cannotexclude the possibility of employing a genomic version of a particulargene where desired.

In certain embodiments, the invention concerns isolated DNA segments andrecombinant vectors which encode a protein or peptide that includeswithin its amino acid sequence an amino acid sequence essentially as setforth in SEQ ID NO:2. Naturally, where the DNA segment or vector encodesa full length MTAP protein, or is intended for use in expressing theMTAP protein, the most preferred sequences are those which areessentially as set forth in SEQ ID NO:2.

The term “a sequence essentially as set forth in SEQ ID NO:2” means thatthe sequence substantially corresponds to a portion of SEQ ID NO:2 andhas relatively few amino acids which are not identical to, or abiologically functional equivalent of, the amino acids of SEQ ID NO:2.The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein, as a gene having a sequenceessentially as set forth in SEQ ID NO:2, and that is associated with atumor suppressor gene. Accordingly, sequences which have between about70% and about 80%; or more preferably, between about 81% and about 90%;or even more preferably, between about 91% and about 99%; of amino acidswhich are identical or functionally equivalent to the amino acids of SEQID NO:2 will be sequences which are “essentially as set forth in SEQ IDNO:2.”

In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:1. The term“essentially as set forth in SEQ ID NO:1,” is used in the same sense asdescribed above and means that the nucleic acid sequence substantiallycorresponds to a portion of SEQ ID NO:1, and has relatively few codonswhich are not identical, or functionally equivalent, to the codons ofSEQ ID NO:1. The term “functionally equivalent codon” is used herein torefer to codons that encode the same amino acid, such as the six codonsfor arginine or serine, as set forth in Table 1, and also refers tocodons that encode biologically equivalent amino acids.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences which may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

Excepting intronic or flanking regions, and allowing for the degeneracyof the genetic code, sequences which have between about 70% and about80%; or more preferably, between about 80% and about 90%; or even morepreferably, between about 90% and about 99%; of nucleotides which areidentical to the nucleotides of SEQ ID NO:1 will be sequences which are“essentially as set forth in SEQ ID NO:1”. Sequences which areessentially the same as those set forth in SEQ ID NO:1 may also befunctionally defined as sequences which are capable of hybridizing to anucleic acid segment containing the complement of SEQ ID NO:1 underrelatively stringent conditions. Suitable relatively stringenthybridization conditions will be well known to those of skill in the artand are clearly set forth herein, for example conditions for use withsouthern and northern blot analysis, and as described in Example 1.

Naturally, the present invention also encompasses DNA segments which arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NO:1. Nucleic acid sequences which are “complementary” arethose which are capable of base-pairing according to the standardWatson-Crick complementarity rules. As used herein, the term“complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1 under relativelystringent conditions such as those described herein.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared whichinclude a short stretch complementary to SEQ ID NO:1, such as about 10to 15 or 20, 30, or 40 or so nucleotides, and which are up to 10,000 or5,000 base pairs in length, with segments of 3,000 being preferred incertain cases. DNA segments with total lengths of about 1,000, 500, 200,100 and about 50 base pairs in length are also contemplated to beuseful.

A preferred embodiment of the present invention is a nucleic acidsegment which comprises at least a 14-nucleotide long stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1. In a more preferred embodiment the nucleic acid is furtherdefined as comprising at least a 20 nucleotide long stretch, a 30nucleotide long stretch, 50 nucleotide long stretch, 100 nucleotide longstretch, a 200 nucleotide long stretch, a 500 nucleotide long stretch, a1000 nucleotide long stretch, or at least an 1118 nucleotide longstretch which corresponds to, or is complementary to, the nucleic acidsequence of SEQ ID NO:1. The nucleic acid segment may be further definedas having the nucleic acid sequence of SEQ ID NO:1.

A related embodiment of the present invention is a nucleic acid segmentwhich comprises at least a 14-nucleotide long stretch which correspondsto, or is complementary to, the nucleic acid sequence of SEQ ID NO:1,further defined as comprising a nucleic acid fragment of up to 10,000 bpin length. A more preferred embodiment if a nucleic acid fragmentcomprising from 14 nucleotides of SEQ ID NO:1 up to 5,000 bp in length,3,000 bp in length, 1,000 bp in length, 500 bp in length, or 100 bp inlength.

Naturally, it will also be understood that this invention is not limitedto the particular nucleic acid and amino acid sequences of SEQ ID NO:1and SEQ ID NO:2. Recombinant vectors and isolated DNA segments maytherefore variously include the MTAP coding regions themselves, codingregions bearing selected alterations or modifications in the basiccoding region, or they may encode larger polypeptides which neverthelessinclude MTAP-coding regions or may encode biologically functionalequivalent proteins or peptides which have variant amino acidssequences.

The DNA segments of the present invention encompass biologicallyfunctional equivalent MTAP proteins and peptides. Such sequences mayarise as a consequence of codon redundancy and functional equivalencywhich are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the MTAP protein or to test MTAPmutants in order to examine tumor suppressor activity or determine thepresence of the MTAP peptide in various cells and tissues at themolecular level.

A preferred embodiment of the present invention is a purifiedcomposition comprising a polypeptide having an amino acid sequence inaccordance with SEQ ID NO:2. The term “purified” as used herein, isintended to refer to a MTAP protein composition, wherein the MTAPprotein is purified to any degree relative to its naturally-obtainablestate, i.e., in this case, relative to its purity within a eukaryoticcell extract. MTAP protein may also be isolated from patient specimens,recombinant cells, tissues, isolated subpopulations of tissues, and thelike, as will be known to those of skill in the art, in light of thepresent disclosure. A purified MTAP protein composition therefore alsorefers to a polypeptide having the amino acid sequence of SEQ ID NO:2,free from the environment in which it may naturally occur.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the MTAP coding regions are aligned within the same expressionunit with other proteins or peptides having desired functions, such asfor purification or immunodetection purposes (e.g., proteins which maybe purified by affinity chromatography and enzyme label coding regions,respectively).

Turning to the expression of the MTAP gene whether from cDNA based orgenomic DNA, one may proceed to prepare an expression system for therecombinant preparation of MTAP protein. The engineering of DNAsegment(s) for expression in a prokaryotic or eukaryotic system may beperformed by techniques generally known to those of skill in recombinantexpression. For example, one may prepare a MTAP-GST(glutathione-S-transferase) fusion protein that is a convenient means ofbacterial expression. However, it is believed that virtually anyexpression system may be employed in the expression of MTAP.

MTAP may be successfully expressed in eukaryotic expression systems,however, it is contemplated that bacterial expression systems can beused for the preparation of MTAP for all purposes. The cDNA containingMTAP may be separately expressed in bacterial systems, with the encodedproteins being expressed as fusions with β-galactosidase, avidin,ubiquitin, Schistosoma japonicum glutathione S-transferase, multiplehistidines, epitope-tags and the like. It is believed that bacterialexpression will ultimately have advantages over eukaryotic expression interms of ease of use and quantity of materials obtained thereby.

It is proposed that transformation of host cells with DNA segmentsencoding MTAP will provide a convenient means for obtaining an MTAPprotein. It is also proposed that cDNA, genomic sequences, andcombinations thereof, are suitable for eukaryotic expression, as thehost cell will, of course, process the genomic transcripts to yieldfunctional mRNA for translation into protein.

Another embodiment is a method of preparing a protein compositioncomprising growing recombinant host cell comprising a vector thatencodes a protein which includes an amino acid sequence in accordancewith SEQ ID NO:2, under conditions permitting nucleic acid expressionand protein production followed by recovering the protein so produced.The host cell, conditions permitting nucleic acid expression, proteinproduction and recovery, will be known to those of skill in the art, inlight of the present disclosure of the MTAP gene.

2.3 Gene Constructs and DNA Segments

As used herein, the terms “gene” and “DNA segment” are both used torefer to a DNA molecule that has been isolated free of total genomic DNAof a particular species. Therefore, a gene or DNA segment encoding atumor suppressor gene refers to a DNA segment that contains sequencesencoding a tumor suppressing protein, but is isolated away from, orpurified free from, total genomic DNA of the species from which the DNAis obtained. Included within the term “DNA segment”, are DNA segmentsand smaller fragments of such segments, and also recombinant vectors,including, for example, plasmids, cosmids, phage, retroviruses,adenoviruses, and the like.

The term “gene” is used for simplicity to refer to a functional proteinor peptide encoding unit As will be understood by those in the art, thisfunctional term includes both genomic sequences and cDNA sequences.“Isolated substantially away from other coding sequences” means that thegene of interest, in this case, a tumor suppressor gene, forms thesignificant part of the coding region of the DNA segment, and that theDNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or cDNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regions,such as sequences encoding leader peptides or targeting sequences, lateradded to the segment by the hand of man.

This invention provides novel ways in which to utilize various knowntumor suppressor DNA segments and recombinant vectors. As describedabove, many such vectors are readily available, one particular detailedexample of a suitable vector for expression in mammalian cells is thatdescribed in U.S. Pat. No. 5,168,050, incorporated herein by reference.However, there is no requirement that a highly purified vector be used,so long as the coding segment employed encodes a tumor suppressorprotein and does not include any coding or regulatory sequences thatwould have a significant adverse effect on suppression of tumor growthand/or cell proliferation. Therefore, it will also be understood thatuseful nucleic acid sequences may include additional residues, such asadditional non-coding sequences flanking either of the 5′ or 3′ portionsof the coding region or may include various internal sequences, i.e.,introns, which are known to occur within genes.

After identifying an appropriate tumor suppressor gene or DNA molecule,it may be inserted into any one of the many vectors currently known inthe art, so that it will direct the expression and production of thetumor suppressor protein when incorporated into a cell. In a recombinantexpression vector, the coding portion of the DNA segment is positionedunder the control of a promoter. The promoter may be in the form of thepromoter which is naturally associated with a tumor suppressor gene, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment or exon, for example, using recombinantcloning and/or PCR™ technology, in connection with the compositionsdisclosed herein.

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a tumor suppressor gene in its naturalenvironment Such promoters may include those normally associated withother tumor suppressor genes, and/or promoters isolated from any otherbacterial, viral, eukaryotic, or mammalian cell. Naturally, it will beimportant to employ a promoter that effectively directs the expressionof the DNA segment in the cell in which the construct is to beintroduced.

The use of recombinant promoters to achieve protein expression isgenerally known to those of skill in the art of molecular biology, forexample, see Sambrook et al. (1989). The promoters employed may beconstitutive, or inducible, and can be used under the appropriateconditions to direct high level or regulated expression of theintroduced DNA segment. The currently preferred promoters are those suchas CMV, RSV LTR, the SV40 promoter alone, and the SV40 promoter incombination with various enhancer elements.

Tumor suppressor genes and DNA segments may also be in the form of a DNAinsert which is located within the genome of a recombinant virus, suchas, for example a recombinant adenovirus, adeno-associated virus (AAV)or retrovirus. In such embodiments, to place the gene in contact with atumor cell, one would prepare the recombinant viral particles, thegenome of which includes the tumor suppressor gene insert, and simplycontact the cells or tissues with the virus, whereby the virus infectsthe cells and transfers the genetic material.

3. BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included tofurther demonstrate certain aspects of the present invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1. PFGE map of the 2.8-Mb YAC contig on 9p21. The individual YAC'sare aligned to the long-range restriction map above each overlappingsegment. Restriction sites are designated as shown. This map does notshow every restriction site for the enzymes SalI, SacII, and SfiIbecause sites that are further away from the probes used were notdetected. The IFNB1 and IFNA genes and pseudogenes and the MTAP, CDKN2,and CDKN2B genes are represented by solid vertical bars. Broad locationsfor the markers D9S736, D9S966, 1063.7, and c1.b are shown. REY and LEYdesignate the right and left YAC vector arms, respectively. YACs A73B12and A88E10 were derived with IFN STS from the St. Louis YAC library(Porterfield et al., 1992). Cosmids and end-rescued plasmid clones arenot shown. Distances are drawn to scale; marked distances are inmegabases.

FIG. 2A. Northern blot of RNA from multiple human tissues hybridizedwith the 3′ 1.4-kb fragment of the MTAP cDNA clone. Lanes: 1, heart: 2,brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8,pancreas.

FIG. 2B. Northern analysis of the blot in FIG. 2A re-probed with β-actincDNA. Lanes: 1, heart: 2, brain; 3, placenta; 4, lung; 5, liver; 6,skeletal muscle; 7, kidney; 8, pancreas.

FIG. 3A. Nucleotide sequence of the MTAP cDNA (SEQ ID NO:1).

FIG. 3B. Protein sequence of the MTAP gene product (SEQ ID NO:2).

FIG. 4. Map of MTAP cDNA clone. Not all restriction sites are indicated.

FIG. 5. Map of the DNA markers on 9p21. The positions of different FISHprobes (YAC11 and COSp16) and molecular markers (M1.4, CDKN2, CDKN2B[p15], and D9S966) are shown.

FIG. 6A. Southern blot analysis of 5 representative cases (lane 1,placental control; lane 2, patient no. 2; lane 3, patient no. 3; lane 4,patient no. 4; lane 5, patient no. 5; lane 6, patient no. 11). Thehybridization is shown with M1.4. Homozygous deletions can be detectedin lanes, 3, 5, and 6. The intensity of the bands in lane 2 issignificantly reduced. The case of lane 4 has a hemizygous 9p deletiondetected by FISH.

FIG. 6B. Southern blot analysis of 5 representative cases (lane 1,placental control; lane 2, patient no. 2; lane 3, patient no. 3; lane 4,patient no. 4, lane 5, patient no. 5; lane 6, patient no. 11). Thehybridization is shown with CDKN2, exon 2. Homozygous deletions can bedetected in lanes, 3, 5, and 6. The intensity of the bands in lane 2 issignificantly reduced. The case of lane 4 has a hemizygous 9p deletiondetected by FISH.

FIG. 6C. Southern blot analysis of 5 representative cases (lane 1,placental control; lane 2, patient no. 2; lane 3, patient no. 3; lane 4,patient no. 4, lane 5, patient no. 5; lane 6, patient no. 11). Thehybridization is shown with CDKN2B, exon 1. Homozygous deletions can bedetected in lanes, 3, 5, and 6. The intensity of the bands in lane 2 issignificantly reduced. The case of lane 4 has a hemizygous 9p deletiondetected by FISH.

FIG. 6D. Southern blot analysis of 5 representative cases (lane 1,placental control; lane 2, patient no. 2; lane 3, patient no. 3; lane 4,patient no. 4, lane 5, patient no. 5; lane 6, patient no. 11). Thehybridization is shown with D9S966. Homozygous deletions can be detectedin lanes, 3, 5, and 6. The intensity of the bands in lane 2 issignificantly reduced. The case of lane 4 has a hemizygous 9p deletiondetected by FISH.

FIG. 6E. Southern blot analysis of 5 representative cases (lane 1,placental control; lane 2, patient no. 2; lane 3, patient no. 3; lane 4,patient no. 4, lane 5, patient no. 5; lane 6, patient no. 11). Thehybridization is shown with the control probe transferrin receptorTFR(E). Homozygous deletions can be detected in lanes, 3, 5, and 6. Theintensity of the bands in lane 2 is significantly reduced. The case oflane 4 has a hemizygous 9p deletion detected by FISH.

FIG. 7A. FISH analysis in 6 cases with the homozygous deletion of theCDKN2 region. The percentage of cells without hybridization signal. (YAC10/2, YAC 11, and COSp16) is shown.

FIG. 7B. FISH analysis in 6 cases with the hemizygous deletions of theCDKN2 region. The percentage of cells with one hybridization signal.(YAC 10/2, YAC 11, and COSp16) is shown. Cases no. 8, 14, 15, and 19have hemizygous deletions of YAC11.

FIG. 8. SSCP analysis of CDKN2, exon 2 with control DNA (lane 1,non-denatured; lane 2, denatured), 2 cases with hemizygous CDKN2deletion (lane 3, no. 1; lane 4, no. 16), and HL60 (lane 5), a cell linewith known point mutation in exon 2. HL60 shows a changed mobility ofbands. None of the patients samples differs from placental DNA.

FIG. 9. 9p deletion map of the cell lines. The position of differentFISH probes (YAC 11, YAC 23, YAC 17, COSp16) and molecular markers(REY24, CDKN2, D9S966, D9S171) are shown. R, homozygous deletions;diagonal lines, non-homozygous deletions, kb, kilobases.

FIG. 10A. FISH analysis in 10 tumor cell lines. The percentage of cellswithout hybridization signal (YAC 10/2, YAC11, COSp16) is shown.

FIG. 10B. FISH analysis in 9 glioblastomas. The percentage of cellswithout hybridization signal (YAC 10/2, YAC 11, COSp16) is shown.

FIG. 11. cDNA map of the MTAP region.

FIG. 12. Southern blot of genomic placental DNA probed with pM1.1. Thelanes are labeled with the corresponding restriction enzyme used.

FIG. 13. Upper panel shows a northern blot of RNA from tumor cell lineshybridized with the 3′ 1.4-kb fragment of the MTAP cDNA clone. The lowerpanel shows the same blot probed with a control cDNA. Lanes 1, HL60;lane 2, HeLa; lane 3, K562; lane 4, Molt 4; lane 5, RAJt; lane 6, SV40;lane 7, A549:7 G361.1.

FIG. 14. Shown is the 34-kDa translation product of the pM1.1 cDNA cloneusing the TNT T3-Coupled Reticulocyte lysate system from Promega Corp.

FIG. 15. Genomic organization of the MTAP gene.

FIG. 16. Titration of M1.1, MLL, and M1.1 and P16 lysates. Shown areabsorbance vs. dilution plots.

FIG. 17A. BV173 Southern Blot Results. From left to right the panelsshow results of the IFNA2 coding region hybridized to HindIII, and EcoRIsupernatant and pellet fractions. Each IFNA fragment containing eachgene member is identified to the right using the proper nomenclature. Rvalues were measured and includes 12 different HindIII, and EcoRI DNAfragments as shown below. These examples were chosen to showsupernatant, and pellet scaffold binding regions. M=λ phage HindIIImarkers. Examples from Southern blots where the coding regions codingplus immediate flanking regions showed 70% or greater supernatantenrichment (R=<30) are a 1.4 kb EcoRI fragment containing IFNA1 (R=<5)(FIG. 17B), and both a 1.2 kb and 1.6 kb EcoRI fragments containingIFNA8 (R=15). Examples where most IFN gene family members mapped to DNAfragments which were enriched greater than 70% in the pellet (R=>70) andwhere it is mapped 5 SARs specifically to either the 5′ or 3′ flankingregions are: a 1-kb EcoRI fragment containing IFNA7 and a 3′ SAR, a58-kb HindIII fragment and a 2-kb EcoRI fragment containing IFNA16 and3′ and 5′ SARs respectively (R=75), a 2.0-kb EcoRI fragment (R=75), anda 4.4-kb HindIII fragment containing IFNA17 and 5′ and 3′ SARsrespectively, an 8.4-kb EcoRI fragment containing IFNA13 and a 5′ SAR, a5.5-kb HindIII fragment containing IFNA14 and a 5′ and 3′ SARs. Exampleswhere additional R values were measured for fragments which containedcoding sequences plus 5′ and 3′ flanking regions and which wereenriched >70% into the pellet fraction: (R=≦70) from the left panel(HindIII) to the right panel (EcoRI), IFNA8 9.4-kb HindIII (R=80),IFNA10 3.5-kb HindIII fragment (R=93). IFNA21 6.0 kb EcoRI (R=86), IFNA24.8-kb EcoRI (R=70), IFNA4, 10, 16, 2.0-kb EcoRI (R=75). These fragmentswere analyzed as containing two strong SARs mapping both 5′ and 3′ tothe coding regions.

FIG. 17B. Strategy used to map SARs in the IFN gene cluster. Restrictionenzyme map of IFNA1 showing the location of SARs both 5′ and 3′ of thegene. See FIG. 17A for representative Southerns. H=HindIII, Bg=BgIII,E=EcoRI, IFNA1→, weak SAR=□, SAR=▪, IFNA2 coding region probe=▪ shownabove IFNA1. R values are represented below the map along with theparticular restriction fragments. A 7.7-kb HindIII fragment (R=80)hybridizing with the IFNA2 coding region was enriched 80% into thepellet (FIG. 17A). The 1.4-kb EcoRI fragment (R=<5) contains the IFNA1coding region and was enriched in the supernatant. Two BglII fragments(each hybridizing to one half of IFNA1), a 1.6-kb fragment (=21) wassupernatant enriched, and a 1.3-kb fragment (R=62) distributed aboutequally into the supernatant and pellet fractions. For this region it ispredicted that two high affinity SARs map outside the coding region pastthe EcoRI sites. The weak binding of the 1.3-kb BglII fragment containspart of the scaffold binding sites from the 5′ high affinity SAR. The 3′SAR begins outside the EcoRI and BglII sites.

FIG. 18. Long-range restriction map of the IFNB1 and IFNA and IFNW genefamilies on Chromosome 9p and the location of SARs. Long-rangerestriction map showing approximately 500 kb of the 9p region containingthe IFNB1, IFNA and IFNW genes. The sizes (in kb) are indicated abovethe restriction map. Restriction enzyme sites are the following:SF=SfiI, S=SalI, Bs=BstI, and n=NotI. All genes and pseudogenes arelabeled with the standard IFN terminology. Long vertical open boxesalong the map represent the IFNB1 coding region the IFNA and IFNPAcoding regions, the IFNW and IFNPW coding regions. Long vertical openboxes also represent the specific pseudogenes. Small black rectangularboxes represent strong SARs, small open boxes represent weak SARs. SARlocations were mapped from the hybridization of the IFNA2 and IFNW1coding regions to SAR (pellet) and non-SAR (supernatant) DNA fractions.

FIG. 19. Restriction enzyme map of the IFNA2, IFNP11, IFN12 and IFNA8gene region showing the location of SARs 1, 2, 3, 4, 5, and 6. In the 3′flanking region of IFNA2 the single high affinity SAR1 begins close tothe gene (approximately 0.5-kb) and then extends 3′ for approximately2.6 kb. The most proximal high affinity IFNA2 SAR2 begins approximately1 kb upstream of the gene and extends 4.8-kb 5′ of the gene. The thirdhigh affinity SAR3 starts approximately 9-kb upstream of IFNA2 andextends 2.5 kb. Weak SARs flank IFNP11 and IFNP12. The size of weak SAR4is 1.4 kb, but the exact sizes of weak SAR5 and SAR6 are unknown.H=HindIII, Bg=BglII, B=BamHI, a–n=restriction DNA fragments used asprobes. →=IFNA2, IFNA8,

=IFNP11, IFNP12, Weak SAR=□, Strong SAR=▪. Horizontal bars below the maprepresent DNA restriction fragments showing different R values. Humangenomic λ clones are shown below map.

FIG. 19B. Southern blot results showing hybridization of probes i, j, k,and l to BglII/BamHI, BamHI/EcoRI, EcoRI and EcoRI/BglII digestedscaffolds. λ HindIII marker lane is represented to the left of theSouthern blot panels. Restriction enzyme map of the non-SAR and SAR3,and SAR4 restriction fragments is represented below and correlates withthe Southern blots. Probe i represents a supernatant enriched DNAfragment whereas probes j, and k (identified above the map) identifypellet enriched fragments of SAR3. Probe 1 identifies a weaker bindingregion just centromeric to SAR3. The 5.7-kb band seen hybridizing withprobe k is most likely the partial EcoRI fragment which contains the0.7-kb EcoRI fragment plus weak SARs 4 and 5. The 3.7-kb band observedin the EcoRI/BglII pellet fraction represents the 3.7-kb EcoRI/EcoRIfragment detecting the most proximal IFNA2 SAR. This result is due toco-purifying the 4.4-kb fragment or probes h, i, and j from the λ 1–3phage. The 4.4-kb EcoRI fragment detects the strong SAR3. See FIG. 19Afor representative R values.

FIG. 20. Restriction enzyme map of SAR3 and SAR4 showing the location ofpellet-enriched regions, weakly bound fragments, andsupernatant-enriched regions. The 1.7-kb RsaI pellet-enriched fragmentoverlaps the previously identified pellet enriched 0.7-kb EcoRI fragment(probe k), thus confirming our earlier mapping of SAR3 (FIG. 19A andFIG. 19B). Supernatant enriched (219-bp HaeIII, 488-bp HaeIII, 390-bpSau3A) and weaker binding fragments (1.0-kb Sau3AI, 1.0-kb HaeIII, and1.14-kb RsaI) defined this region further into non-SAR fragments and theweak SAR4 respectively. Because the 488-bp HaeIII fragment (supernatantenriched) overlapped the telomeric end of the 1.0-kb Sau3A fragment by300 bp, the remaining centromeric Sau3A. 700 bp should contain scaffoldbinding sites responsible for the weak binding pattern. In addition, the1.14-kb RsaI fragment, and the 1.0-kb HaeIII appeared to bind weakly toscaffold proteins supporting the conclusion that weak binding sites mapto the region of overlap between all three weak binding fragments (1.14kb RsaI, 1.0 kb HaeIII, and 1.0 kb Sau3A. Bam=BamHI, Sau=Sau3A,Rsa=RsaI, Hae=HaeIII, Bgl=BglII. Restriction fragments hybridized toprobe 1 (3.0-kb EcoRI/BglII) are indicated in region below restrictionmap and SAR. R values are represented under the restriction fragments.Strong SAR=▪ Weak SAR=□.

FIG. 21. U373 Southern blot results. From left to right the panels showresults of the IFNA2 coding region hybridized to HindIII, and EcoRIsupernatant and pellet fractions. Each IFNA fragment containing eachgene member is identified to the right using the proper nomenclature. Rvalues were measured and includes 16 different HindIII, EcoRI and BglIIDNA fragments. These examples were chosen as representative examplesshowing supernatant, pellet and weak scaffold binding regions. M=λ phageHindIII markers. Some examples are the following: strong SARs on HindIIIfragments (molecular weights are the same as in BV173), containing IFNA8(R=71), IFNA4,6,10 (R=74); non-SARs with negligible binding containingIFNA1,8 (R=23), and IFNA7 (R=21).

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

4.1 CDKN2

Even though there is great controversy regarding the role of CDKN2 inmultiple cancers except melanoma, it is agreed that the 9p chromosomeregion harbors important gene(s) relevant to cancer because it isfrequently deleted in cancer cells. The definition of the geneticlesions involved in oncogenesis has advanced through the identificationof multiple dominant oncogenes and tumor suppressor genes. The presentinvention concerns in a general sense alterations in CDKN2, a geneinvolved in cell cycle control and any other 9p genes that maycontribute to the malignant process in gliomas and other tumor types. Inaddition to improving the understanding of the basic mechanisms involvedin gliomagenesis, the present invention also concerns improvedstrategies for prevention, diagnosis and treatment of this uniformlyfatal cancer.

4.2 Human Gene Encoding MTAP

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP)enzyme activity. The gene encoding this enzyme was previously mapped tothe short arm of chromosome 9, band p21–22, a region that is frequentlydeleted in multiple tumor types. To clone candidate tumor suppressorgenes (TSGs) from the deleted region on 9p21–22, a long range physicalmap of 2.8 megabases on 9p21 has been constructed using overlappingyeast artificial chromosome (YAC) and cosmid clones. This map includesthe type I IFN gene cluster, the recently identified candidate TSGs,CDKN2 (p16^(INK4A)) and CDKN2B (p15^(INK4B)) as well as several CpGislands. In addition, other transcription units have been identifiedwithin the YAC contig.

Sequence analysis of a 2.5 kb cDNA clone isolated from a CpG island thatmaps between the IFN genes and CDKN2 reveals a predicted open readingframe of 283 amino acids followed by 1302 nucleotides of 3′ untranslatedsequence. This gene is evolutionarily conserved and shows significantamino acid homologies to mouse and human purine nucleosidephosphorylases as well as to a hypothetical 25.8 kDa protein in the petgenes (coding for cytochrome bc₁-complex) region of Rhodospirillumrubrum. The location, expression pattern and nucleotide sequence of thisgene suggest that it codes for the MTAP enzyme.

4.3 Genetic Deletions in the 9p21 Region

Deletions of chromosomal band 9p21 have been detected in various tumortypes as well as in more than 20% of acute lymphoblastic leukemia (ALL).These deletions frequently include the entire interferon (IFN) genecluster as well as the methylthioadenosine phosphorylase (MTAP) gene.Recently, the CDKN2 gene p16^(INK4A), MTS I, CDK4I) has been proposed asa candidate tumor suppressor gene on 9p21 because it is frequentlydeleted in cell lines derived from multiple tumor types. To determine ifCDKN2 or another closely related gene on 9p is the target of 9pdeletions in ALL and other hematological malignancies, 20 patientsamples (13 ALL, 2 AML, 5 NHL) with 9p rearrangements were examined bySouthern blot analysis, fluorescence in situ hybridization (FISH) andsingle-strand conformation polymorphism (SSCP) for alterations of CDKN2.

Homozygous deletions of the CDKN2 region were detected in 10 cases(50%): 6 ALL, 2 AML, 2 NHL. In one additional case the intensity of theSouthern blot band was significantly reduced suggesting a CDKN2 deletionin a subpopulation of the malignant cells. The interferon (IFN) genecluster was homozygously deleted in 2 of 15 (13%) analyzed cases, MTAPwas deleted in 6 of 15 analyzed cases (40%). In addition, hemizygousdeletions of the CDKN2 region were identified in 6 ALL cases byinterphase FISH. No point mutation of the coding region of CDKN2 wasdetected by SSCP in the 6 cases with hemizygous deletions. It wasconcluded that CDKN2 is the most frequently homozygously deleted markeron 9p. The absence of point mutations in the coding region of CDKN2 incases with hemizygous 9p deletions and the frequent co-deletion of MTAPand other yet unidentified neighboring genes suggest that thesimultaneous deletion of these genes may be responsible for theselective growth advantage for the malignant cells.

4.4 Brain Tumors

Gliomas (a spectrum of tumors ranging from well differentiated low gradeastrocytomas to high grade glioblastoma multiforme) constitute about 60%of all primary brain tumors. Brain tumors are the most common group ofsolid tumors of childhood, occurring in two age peaks; 3 yrs–12 yrs and50 yrs–70 yrs (Díaz et al., 1990). Patients with low grade gliomas areusually curable while patients with glioblastoma multiforme have amedian survival of less than 1 year even with the best conventionaltherapy of resection, followed by radiation therapy and chemotherapy.There is currently no uniform way of classifying these tumors ordetermining which tumors will progress rapidly. The lack of progress indiagnosing and treating this neoplasm is due in part to the lack ofunderstanding of their basic biology. There is little understanding ofthe etiology, mechanisms responsible for tumor progression and responseto therapy. Recent work on the molecular characterization of braintumors highlight the importance of accumulation of genetic alterationsin the progression of these tumors. Comparison of molecular, genetic andhistopathologic analysis of glial tumors suggests specific associationsbetween histologic types and genetic alterations. However, there iscurrently only a small database with which to evaluate suchrelationships. By studying these tumors at the molecular level, one maybetter understand their biology, which ultimately will lead to improveddiagnosis and management of patients with gliomas and other cancers.Except for TP53 Cancer arises as a result of the accumulation of aseries of genetic mutations which lead to progressive disorganization ofthe control mechanisms that normally limit cell growth and proliferationNational Center for Health Statistics, 1987).

Cytogenetic analysis of solid tumors has proven to be relativelydifficult. Nonetheless, many of the genes cloned to date that areimportant in solid tumor progression have been isolated because theirlocation was defined by recurring chromosome aberrations in theparticular tumors or by linkage analysis in familial cases. Recentstudies highlight the importance of tumor suppressor genes, includingTP53, and RB1, in the development of solid tumors (Bigner et al., 1988;Kacker et al., 1990; Lukeis et al., 1990; Allegra, 1992; Cowan andFrancke, 1992; Petty et al., 1993). Work has confirmed the view thatcarcinogenesis is a multistep process. In colon cancer, a number of theinvolved genes have now been cloned, including DCC, APC, MSH2, and MLH1(Kacker et al., 1990; Lukeis et al., 1990).

There is paucity of information in the cytogenetic database on solidtumors but recent data indicate that unbalanced translocations ordeletions of 9p with the shortest region of overlap (SRO) at 9p22 arerecurring abnormalities in a gliomas as well as other tumor types suchas melanoma, non-small cell lung cancer, head and neck cancer,mesothelioma, ovarian and breast cancers. By molecular analysis,homozygous deletions of DNA sequences on 9p or loss of heterozygosityhas been described in a significant percentage of these tumors. Themolecular studies in the different tumor types have demonstrated thatthese deletions involving 9p are sometimes submicroscopic and ofteninclude homozygous deletions of all or part of the interferon (IFN) genecluster and the methylthioadenosine phosphorylase (MTAP) gene. Both theIFN gene cluster and MTAP gene have been mapped at 9p21–p22. Thefrequent occurrence of homozygous deletions in this chromosomal regionstrongly suggested the presence of a tumor suppressor gene (TSG), whoseinactivation is involved in the diverse types of tumors. In addition,Cannon-Albright et al. reported on the linkage of a subset of familialmelanoma to this locus on chromosome 9p (MLM), the same region that hasbeen shown to be deleted in melanoma cell lines and primary tumors. Itis proposed that this TSG(s) is likely to be important in the moregeneral pathway of oncogenesis.

The introduction of a TSG (or a single normal chromosome carrying such agene) into tumorigenic cells or the use of inter-specific and morerecently, intra-specific human somatic cell hybrids provides afunctional assay for the presence of TSGs (Díaz et al., 1990; Petty etal., 1993). Suggestion that there might be tumor suppressor activity onhuman chromosome 9 has been in the literature from as far back as 1975.Earlier work with somatic cell hybrids between mouse neoplastic cellsand normal human fibroblasts, which segregate human chromosomes, showedthat the human chromosome 9 is preferentially lost from tumorigenichybrids, possibly indicating that hybrid cells which contain a normalhuman chromosome 9 can not form tumors in nude mice (Deville et al.,1991). A more recent report has also shown that after the introductionof human chromosomes 1, 6, 9, 11, and 19, only chromosomes 1, 6, and 9were able to completely suppress tumorigenicity in a uterine endometrialadenocarcinoma cell line (Bieche et al., 1992). These studies supportthe hypothesis that tumor suppressor activity is present on humanchromosome 9 and functions in a mouse background. The gene is alsolikely to function as a TSG in rat and mouse because tumor suppressorfunction have been previously mapped to rat chromosome 5 and mousechromosome 4 in the region of synteny to human chromosome 9p, which mapsaround the IFN locus. The working hypothesis has been that the TSG(s) isclosely linked to the IFN and MTAP genes on 9p (since retention of IFNgenes and MTAP were sometimes observed despite deletion of DNA sequenceson 9p) and that these genes are included in the deletions only as“innocent bystanders”. One could not, however, completely exclude theIFN genes because of their negative role in growth regulation andsuggested that they may play an accessory role in tumor suppression bythis locus on 9p.

4.5 Regulation of Cell Proliferation

Regulation of cell proliferation appears to be a complex processinvolving the regulated expression and/or modification of discreet geneproducts, including that of inhibitory growth regulators such assecretory proteins like interferons and nuclear phosphoproteins like RB.Interferons inhibit cell proliferation and in many cases this inhibitionis mediated by an autocrine pathway (Díaz et al., 1988). It has beenproposed that IFN may participate in a feedback mechanism to regulatecell proliferation in adult tissues. Interferon induces RB expression inSW 480 (IFN transduced) colon cancer cell line but not in Daudi growthresistant and DU-145 RB(-) cell line. Interferons have also been shownto down-regulate MYC expression which is thought to be mediated by RBprotein interaction with the MYC promoter in Daudi cells. Interferon hasalso been shown to down regulate SRC and RAS in RT4 bladder cancer cellline. Moreover, the interferon inducible proteins implicated in tumorsuppression include the interferon regulatory factors, IRF-1 and IRF-2,a double stranded RNA-activatable protein kinase and RNase L, a latentendoribonuclease to mention a few. The deletion of IFN genes maytherefore lead to the deregulation of cell proliferation, giving rise toclonal expansion of the mutated cell.

MTAP on the other hand is a gene that codes for an enzyme involved inthe metabolism of polyamines and purines. This enzyme is present in allnormal tissues and in cell lines derived from normal cells but isdeficient in cell lines established from leukemias, lymphomas, and solidtumors such as melanoma, breast cancer, squamous cell lung cancer andrectal adenocarcinoma. In mammalian cells, methylthioadenosine (MTA),the substrate for MTAP is produced during synthesis of polyamines fromdecarboxylated S-adenosylmethionine. MTA does not accumulate in normaltissues but is cleaved rapidly to adenine and 5′-methylthioribose1-phosphate (MTR-1-P) by MTAP. The adenine is recycled to purinenucleotides via adenine phosphoribosyltransferase. MTAP deficiency, bydecreasing adenine formation, would be expected to interfere with thissalvage pathway by decreasing adenine formation. On the other hand,MTR-1-P is converted to methionine, which may also be synthesized fromhomocysteine by methionine synthase and betaine-homocysteinemethyltransferase. In MTAP deficient cells, however, methionine issynthesized solely from homocysteine. Accordingly, MTAP deficientmalignant cells might become more dependent than normal cells on anexogenous supply of methionine. Thus, MTAP deficiency in humanmalignancy may permit the development of enzyme-selective chemotherapyagents in which enzyme-negative cancer cells will be killed with drugscausing the depletion of purine nucleotides or methionine, underconditions in which enzyme-positive normal cells can be rescued bygiving MTA as a source of purines or methionine. This major differencebetween normal and malignant cells might be used to design moreeffective chemotherapy approaches in gliomas and other solid tumorswhere there are currently no effective therapy.

4.6 Western Blots

The compositions of the present invention will find great use inimmunoblot or western blot analysis. Anti-MTAP antibodies may be used ashigh-affinity primary reagents for the identification of proteinsimmobilized onto a solid support matrix, such as nitrocellulose, nylonor combinations thereof. In conjunction with immunoprecipitation,followed by gel electrophoresis, these may be used as a single stepreagent for use in detecting antigens against which secondary reagentsused in the detection of the antigen cause an adverse background. Thisis especially useful when the antigens studied are immunoglobulins(precluding the use of immunoglobulins binding bacterial cell wallcomponents), the antigens studied cross-react with the detecting agent,or they migrate at the same relative molecular weight as across-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

4.7 Vaccines

The present invention contemplates vaccines for use in both active andpassive immunization embodiments. Immunogenic compositions, proposed tobe suitable for use as a vaccine, may be prepared most readily directlyfrom immunogenic MTAP peptides prepared in a manner disclosed herein.Preferably the antigenic material is extensively dialyzed to removeundesired small molecular weight molecules and/or lyophilized for moreready formulation into a desired vehicle.

The preparation of vaccines which contain MTAP peptide sequences asactive ingredients is generally well understood in the art, asexemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;4,599,230; 4,596,792; and 4,578,770, all incorporated herein byreference. Typically, such vaccines are prepared as injectables. Eitheras liquid solutions or suspensions: solid forms suitable for solutionin, or suspension in, liquid prior to injection may also be prepared.The preparation may also be emulsified. The active immunogenicingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient Suitable excipientsare, for example, water, saline, dextrose, glycerol, ethanol, or thelike and combinations thereof. In addition, if desired, the vaccine maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents, or adjuvants which enhance theeffectiveness of the vaccines.

Vaccines may be conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyallalene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient in the range ofabout 0.5% to about 10%, preferably about 1 to about 2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain about 10to about 95% of active ingredient, preferably about 25 to about 70%.

The MTAP and MTAP-derived peptides of the present invention may beformulated into the vaccine as neutral or salt forms.Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the peptide) and those which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges areof the order of several hundred micrograms active ingredient pervaccination. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection or the like. The dosage of the vaccine willdepend on the route of administration and will vary according to thesize of the host.

Various methods of achieving adjuvant effect for the vaccine includesuse of agents such as aluminum hydroxide or phosphate (alum), commonlyused as about 0.05 to about 0.1% solution in phosphate buffered saline,admixture with synthetic polymers of sugars (Carbopol®) used as an about0.25% solution, aggregation of the protein in the vaccine by heattreatment with temperatures ranging between about 70° to about 101° C.for a 30-second to 2-minute period, respectively. Aggregation byreactivating with pepsin treated (Fab) antibodies to albumin, mixturewith bacterial cells such as C. parvum or endotoxins orlipopolysaccharide components of Gram-negative bacteria, emulsion inphysiologically acceptable oil vehicles such as mannide mono-oleate(Aracel-A®) or emulsion with a 20% solution of a perfluorocarbon(Fluosol-DA®) used as a block substitute may also be employed.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1–5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the supernatant antigens. The assays may be performed bylabeling with conventional labels, such as radionuclides, enzymes,fluorescents, and the like. These techniques are well known and may befound in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932;4,174,384 and 3,949,064, as illustrative of these types of assays.

4.8 DNA Segments

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a MTAP peptide in itsnatural environment. Such promoters may include promoters normallyassociated with other genes, and/or promoters isolated from any viral,prokaryotic (e.g., bacterial), eukaryotic (e.g., fungal, yeast, plant,or animal) cell, and particularly those of mammalian cells. Naturally,it will be important to employ a promoter that effectively directs theexpression of the DNA segment in the cell type, organism, or evenanimal, chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology, for example, see Sambrook et al., 1989.The promoters employed may be constitutive, or inducible, and can beused under the appropriate conditions to direct high level expression ofthe introduced DNA segment, such as is advantageous m the large-scaleproduction of recombinant proteins or peptides. Appropriatepromoter/expression systems contemplated for use in high-levelexpression include, but are not limited to, the Pichia expression vectorsystem (Pharmacia LKB Biotechnology), a baculovirus system forexpression in insect cells, or any suitable yeast or bacterialexpression system.

In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of MTAP peptides orepitopic core regions, such as may be used to generate anti-MTAPantibodies, also falls within the scope of the invention. DNA segmentsthat encode MTAP peptide antigens from about 10 to about 100 amino acidsin length, or more preferably, from about 20 to about 80 amino acids inlength, or even more preferably, from about 30 to about 70 amino acidsin length are contemplated to be particularly useful.

In addition to their use in directing the expression of MTAP peptides ofthe present invention, the nucleic acid sequences contemplated hereinalso have a variety of other uses. For example, they also have utilityas probes or primers in nucleic acid hybridization embodiments. As such,it is contemplated that nucleic acid segments that comprise a sequenceregion that consists of at least an about 14-nucleotide long contiguoussequence that has the same sequence as, or is complementary to, an about14-nucleotide long contiguous DNA segment of SEQ ID NO:1 will findparticular utility. Longer contiguous identical or complementarysequences, e.g., those of about 20, 30, 40, 50, 100, 200, 300, 500,1000, (including all intermediate lengths) and even those up to andincluding about 1118-bp (full-length) sequences will also be of use incertain embodiments.

The ability of such nucleic acid probes to specifically hybridize toMTAP-encoding sequences will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are envisioned, including the use of the sequence information forthe preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of about 14, 15–20, 30, 40, 50, or even of about100 to about 200 nucleotides or so, identical or complementary to theDNA sequence of SEQ ID NO:1, are particularly contemplated ashybridization probes for use in, e.g., Southern and Northern blotting.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 10–14 and up to about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

The use of a hybridization probe of about 14 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 14 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of about 15 to about 20 contiguousnucleotides, or even longer where desired.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as PCR™, by introducing selected sequences intorecombinant vectors for recombinant production, and by other recombinantDNA techniques generally known to those of skill in the art of molecularbiology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C. Such selective conditions tolerate little,if any, mismatch between the probe and the template or target strand,and would be particularly suitable for isolating MTAP-encoding DNAsegments. Detection of DNA segments via hybridization is well-known tothose of skill in the art, and the teachings of U.S. Pat. Nos. 4,965,188and 5,176,995 (each incorporated herein by reference) are exemplary ofthe methods of hybridization analyses. Teachings such as those found inthe texts of Segal, 1976; Prokop, 1991; Kuby, 1994; and Maloy et al.,1994, are particularly relevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate MTAP-encodingsequences from related species, functional equivalents, or the like,less stringent hybridization conditions will typically be needed inorder to allow formation of the heteroduplex. In these circumstances,one may desire to employ conditions such as about 0.15 M to about 0.9 Msalt, at temperatures ranging from about 20° C. to about 55° C.Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmental undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

4.9 Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thedata shown in Table 1.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CCA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W USGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art byte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

4.10 Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, for example, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA. Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.

4.11 Monoclonal Antibodies

Means for preparing and characterizing antibodies are well known in theart (See, e.g., Harlow and Lane, 1988; incorporated herein byreference).

The methods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies;Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified MTAP or MTAP-related protein, polypeptide or peptide.The immunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep frog cells is alsopossible. The use of rats may provide certain advantages (Goding, 1986),but mice are preferred, with the BALB/c mouse being most preferred asthis is most routinely used and generally gives a higher percentage ofstable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Campbell, 1984; Goding, 1986). For example, wherethe immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (vol/vol) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot inimunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

4.12 Pharmaceutical Compositions

The pharmaceutical compositions disclosed herein may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or they may be enclosed in hard or soft shell gelatincapsule, or they may be compressed into tablets, or they may beincorporated directly with the food of the diet. For oral therapeuticadministration, the active compounds may be incorporated with excipientsand used in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations should contain at least 0.1% of activecompound. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 2 to about 60%of the weight of the unit. The amount of active compounds in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fingi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial ad antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-dying and freeze-dryingtechniques which yield a powder of the active ingredient plus nyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral prophylaxis the polypeptide may be incorporated with excipientsand used in the form of non-ingestible mouthwashes and dentifrices. Amouthwash may be prepared incorporating the active ingredient in therequired amount in an appropriate solvent, such as a sodium boratesolution (Dobell's Solution). Alternatively, the active ingredient maybe incorporated into an antiseptic wash containing sodium borate,glycerin and potassium bicarbonate. The active ingredient may also bedispersed in dentifrices, including: gels, pastes, powders and slurries.The active ingredient may be added in a therapeutically effective amountto a paste dentifrice that may include water, binders, abrasives,flavoring agents, foaming agents, and humectants.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

The composition can be formulated in a neutral or salt form.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035–1038 and 1570–1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques and methods contemplated to function well in the practice ofthe invention, and thus can be considered to constitute preferred modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

5. EXAMPLES 5.1 Example 1 Construction of a 2.8 Mb YAC Contig andCloning of the Human MTAP Gene from the Tumor Suppressor Region on 9p21

This example describe the construction of a long range physical maparound the IFN gene cluster which covers a distance of 2.8 Mb asdetermined by pulsed-field gel electrophoresis and also the isolation ofthe MTAP gene cDNA. All of the known genes were localized as well asseveral CpG islands on this map. Restriction sites and pulsed fieldfragment sizes are clearly delineated on the resultant map which extendsfurther proximally than the one previously presented (Weaver-Feldhaus etal., 1994) In addition, several new markers are described and localizedon this map. The approximate location of the shortest region of overlapof 9p deletions in gliomas, melanomas, lung cancer, leukemia,mesothelioma, head and neck cancer and bladder cancers in relation tothis map is indicated.

5.1.1 Materials and Methods

5.1.1.1 Cell Lines

The clinical and cytogenetic characterization of the tumor cell linesused in this study have been previously published (Olopade et al., 1992;1993). Cell lines were used for the deletion mapping because theyprovide an indefinite supply of DNA and because it had previously beenshown that the deletions present in cell lines were similar to thedeletions observed in primary leukemias and gliomas. (Díaz et al., 1988;1990; Olopade et al., 1992).

5.1.1.2 Analysis of YAC Clones

YAC clones corresponding to IFNA1 (Kwiatkowski and Díaz, 1992; Díaz etal., 1994) and D9S966 (Bohlander et al., 1994) STSs were isolated fromthe CEPH MegaYAC library. High-molecular-weight YAC DNA was isolateddigested, electrophoresed and blotted as previously described (Díaz etal., 1994). To detect the left and right YAC vector arms, a 346 bpHindIII-BamHI and a 276-bp BamHI-SalI restriction fragment from theplasmid pBR322 were used respectively. Other probes used are asdescribed in FIG. 1. The DNA probes were labeled with [α-³²P]dATP usingthe random primer labeling technique (Feinberg and Vogelstein, 1983).PCR™ of the STSs was performed with 50 ng of YAC or human genomic DNA astemplate using standard PCR™ conditions.

5.1.1.3 Fluorescence in Situ Hybridization (FISH) Analysis

YAC clones were purified by pulsed field gel electrophoresis and the DNAwas amplified using a sequence-independent amplification technique(Bohlander et al., 1994). The amplification products were then labeledwith biotin-11-dUTP, or directly labeled nucleotides (spectrum-orange™,VYSIS), and FISH was performed as previously described (Rowley et al.,1990).

5.1.1.4 Cloning YAC End-Specific Clones

To obtain YAC end specific probes, YAC end rescue was performed asdescribed (Hermanson et al., 1991). Single copy fragments from the YACend rescued inserts were used as probes for Southern hybridization toDNA of the different YACs as well as to a panel of tumor cell lines.Each end clone probe was also used to screen a copy of the LawrenceLivermore Laboratory chromosome 9 flow-sorted cosmid library. Theoverlap between YACs was identified by comparing Southern blots afterhybridization to the IFNA2 gene probe which cross hybridizes to a largenumber of IFN genes, and after hybridization to the different end cloneprobes. YACs were also aligned by comparison of their long rangerestriction maps.

5.1.1.5 Screening for Expressed Sequences

The cosmids obtained using probes from this ordered YAC contig were usedfor several different strategies to detect expressed sequences includingdirect screening of cDNA libraries, exon trapping, and a cDNA selectionprotocol based on the capture of sequence independently amplified cosmidfragments by biotinylated cDNA (Buckler et al., 1991; Lovett et al.,1991). Exon trapping was performed according to the manufacturer'sprotocol using the Exon Trapping kit (Life Technologies, Inc.,Gaithersburg, Md.). Each exon-trapped product or cDNA selected fragmentfrom the cosmids was hybridized to a multiple tissue Northern blot(Clontech, Palo Alto, Calif.), a somatic cell hybrid panel (Oncor,Gaithersburg, Md.), “zoo blots” (Bios, New Haven, Conn.) and Southernblots of tumor cell lines. Products that detected transcripts onNorthern analysis were used to screen cDNA libraries from human adultbrain, fetal brain and fibroblast constructed in λgt10 or λgt11(Clontech). In each case, approximately 7.5×10⁵ plaques were screened.Positive clones were subcloned into the pBluescript vector (Stratagene,La Jolla, Calif.) for sequencing.

5.1.1.6 Nucleotide Sequencing and Generation of STSs

Sequencing was performed on an ABI model 373A DNA sequencing system withthe PRISM Ready Reaction Dye-Deoxy Terminator Cycle sequencing kit(Applied Biosystems). cDNA clones were sequenced entirely on bothstrands using double stranded templates. The DNA sequence and thepredicted open reading frame were compared with GenBank databases byusing the BLASTN and BLASTP programs (Altschul et al., 1990).

5.1.2 Results

5.1.2.1 Construction of the Physical Map

Fifteen YAC clones were identified after screening the CEPH MegaYAClibrary with the IFNA and D9S966 STSs. Five clones (33%) were found tobe non chimeric by FISH analysis and were further analyzed. Two YACclones (YAC 802B11 and 886F9) contain the entire IFN gene clusterwhereas the remaining three YAC clones (883G5, 942A3, and 807E4) containD9S966. YACs A73B12 and A88E10 were obtained with consensus IFN STS fromthe St Louis YAC Library (Díaz et al., 1994). The YACs were digestedwith the rare cutting restriction endonucleases NotI, SacII, SalI, andSfiI. After pulsed-field gel electrophoresis, and Southern transfer, theblots were hybridized to a battery of probes including the IFNA, IFNB1,the left and right vector arm probes, D9S966, six end probes and theCDKN2 cDNA probe. The resulting map is shown in FIG. 1. The IFN genescontained within YACs 802B11 and 886F9 were identified and aligned withthe previous map of the IFN gene cluster (Díaz et al., 1994). Except forYAC 886F9, none of the YACs demonstrated any unusual deletions orrearrangements as determined by STS content. The 886F9 YAC publishedpreviously (Weaver-Feldhaus et al., 1994) is larger and extends furthercentromeric than the clone isolated, suggesting that this YAC may haveundergone an internal deletion. However, the STS content of theremaining human insert was consistent with the other IFN-derived YACs.To characterize the YAC clones further, single copy DNA fragmentsobtained from the YAC end clones were used as probes on Southernhybridization. The results are included in FIG. 1. Each end clone probemapped back to the respective YACs and to chromosome 9 by FISH analysis.This map does not show every restriction site for the enzymes SalI,SacII, and SfiI because sites that are further away from the probes usedwere not detected. However, several CpG islands can readily beidentified on this map.

5.1.2.2 Deletion Mapping Analysis

Each unique fragment from the end-clones and additional STSs were testedon a panel of cell lines to refine the deletion map. The results aresummarized in Table 2. Homozygous deletion of at least one markerderived from this YAC contig was detected in 69% of glioma cell lines,45% of melanoma cell lines, 50% of bladder cancer cell lines, 89% ofleukemias, 100% of mesotheliomas, 38% of head and neck cancer and 34% oflung cancer cell lines. The majority of the cell lines had largehomozygous deletions which overlapped around CDKN2/CDKN2B.

It had previously been shown that the deletion in Hs294T, a melanomacell line could not be complemented by introducing a chromosome 9derived from the T98G cell line by microcell chromosome transfer.However, introducing a normal short arm of chromosome 9 derived from ahuman fibroblast cell line induced senescence in Hs294T (Porterfield etal., 1992). The region deleted in Hs294T is flanked by D9S736 andD9S966. In T98G, the homozygous deletion is flanked by MTAP and CDKN2B.Therefore, a region of approximately 100 kb was defined by theoverlapping homozygous deletions in these two cell lines. Thus, it waspossible to define a shortest region of overlap (SRO) of these 9pdeletions to the region between the 3′ end of MTAP and CDKN2B. FromTable 2, it is apparent that the pattern and percentage of 9p homozygousdeletions differ in different tumor types. For example, in melanomas,mesotheliomas and head and neck cancers, the deletions rarely extendinto the IFN gene cluster, whereas the IFN genes are included in 27–44%of the deletions in leukemias, bladder cancer and gliomas. Moreover,MTAP is homozygously deleted with the same frequency as CDKN2 in sometumor types.

TABLE 2 Homozygous Loss of 9p Markers in Tumor Cell Lines % cell linestest showing homozygous deletions Cell type (n) IFNB1 IFNA D9S736 M1.4CDKN2 CDKN2B D9S966 D9S171 Leukemia (18) 39 44 ND 65 89 78 44 6 Melanoma(18) 0 0 0 ND 45 45 15 0 Glioma (26) 27 42 42 63 69 65 42 12 Bladder(16) 0 31 ND 50 50 50 44 ND Head and neck (8) 0 0 0 0 38 25 0 ND Lung(58) 6 8 ND 34 34 29 5 2 Mesothelioma (5) 0 0 0 100 100 100 40 20Homozygous deletion of these markers were detected by Southern blotanalysis or STS-PCR ™. The location of the markers are shown in FIG. 1.ND, not done; M1.4, 1.4-kb fragment from the 3′ untranslated portion ofthe MTAP gene.

The following markers were not present in the YAC contig: D9S3, D9S126,D9S171, D9S162, D9S962 (MDS10), D9S963 (MDS36), and an STS from theD9S171 YAC which maps at least 500 kb telomeric of D9S171. It waspossible to localize D9S736 within YACs 802B11 and YAC 886F9 in a 170 kbSalI-SfiI fragment centromeric to the IFN gene cluster and close to theright end of YAC 886F9; 1063.7 was present in YAC 807E4 only and c1.b inYACs 942A3 and 807E4. (FIG. 1) (Kamb et al., 1994; Weaver-Feldhaus etal., 1994). Because the distance from the IFN gene cluster to thecentromeric end of this YAC contig is 1.8 Mb, D9S171 should be a minimumdistance of 2.3 Mb from the centromeric end of the IFN gene cluster; andD9S736 should be at least 2.0 Mb from D9S171. This is consistent withprevious estimates. D9S736 has been estimated to be 2 cM from D9S171(Weaver-Feldhaus et al., 1994), whereas D9S126 was estimated to be at aminimum distance of 1.0 Mb from the IFN gene cluster (Fountain et al.,1992).

5.1.2.3 Expressed Sequences within and Around the SRO

An 85-bp exon trapped product obtained from a cosmid which maps in theCpG island at the right end of YAC 886F9 was used to screen a cDNAlibrary. One of the clones, a 2.5-kb cDNA detects 2 major transcripts,of about 2.3 kb and 6.0 kb as shown in FIG. 2A and FIG. 2B. This gene isexpressed to varying degrees in all tissue types and is conserved in allmammalian species as judged by zoo-blot hybridization. The nucleotidesequence (FIG. 3A) (SEQ ID NO:1) reveals an open reading frame (ORF)coding for 283 amino acids (FIG. 3B) (SEQ ID NO:2) which included theinitiator methionine codon. The protein sequence shows homology to thehuman, mouse, and bacteria purine nucleoside phosphorylase gene (PNP),as well as to a hypothetical 25.8 kDa protein in the pet genes (codingfor cytochrome bc₁-complex) region of Rhodospirillum rubrum, and also toa recently described ORF from S. cerevisiae. MTAP is a PNP but hasdifferent substrate specificity than the PNPs that have been cloned atpresent. The region of homology to the 25.8 kDa protein is distinct fromthe region of homology to the purine nucleoside phosphorylases with onlya minor overlap.

The presence or absence of a 1.4 kb subclone of the cDNA (probe M1.4,FIG. 4) was correlated with the presence or absence of MTAP enzymeactivity in previously characterized cell lines (Olopade et al., 1992;1993). This 1.4-kb probe is deleted in every cell line which lack noMTAP enzyme activity and is present in all cell lines with MTAP enzymeactivity. When the 1.1-kb, 5′ fragment of the cDNA probe (M1.1, FIG. 4)was used for Southern blot analysis of PstI digested human genomic DNA,four bands were seen. One of these bands was always seen in cell lineswith homozygous deletions of the 9p21 region. The M1.1 probe was thenused on a somatic cell hybrid panel, and found to hybridize only tohuman chromosomes 9 and 3. Thus, it appears that another gene orpseudogene homologous to MTAP maps to human chromosome 3.

5.1.3 Discussion

The MTAP gene was previously localized using somatic cell hybrids(Carerra et al., 1984). The location was refined using informationobtained by performing pulsed field gel electrophoresis in cell lines(Díaz et al., 1988; Olopade et al., 1992). Because there was no probeavailable for the MTAP gene, it was concluded that the putative TSG mustlie between the IFN gene cluster and the MTAP gene. Using similarreasoning, Coleman et al. (1994) placed the SRO in melanomas centromericto the MTAP gene and suggested that the SRO in melanoma was distinctfrom the SRO in gliomas, leukemias and lung cancers. Barring any complexrearrangements in both T98G (glioma) and Hs294T (melanoma), the positionof the TSG should be within the region defined by the homozygousdeletions in these two cell lines. This region maps centromeric to M TAPand the IFN gene cluster but distal to D9S966 and includes CDKN2 (FIG.1). This region corresponds to the only critical region defined usingprimary samples from patients with gliomas and leukemias (Dreyling etal., 1995). The region overlaps the MLM locus because it maps in the 2cM region between D9S736 and D9S171 (Cannon-Albright et al., 1984; Poveyet al., 1994). These data are consistent with recent published results(Jen et al., 1994) in which a high frequency of homozygous deletions ofCDKN2 and CDKN2B was found in primary glioma samples. No point mutationsof either gene were observed in primary gliomas.

The long range map covers 2.8 megabases including the IFN gene locus butdoes not reach D9S126 or D9S171. There are now 2 reports of homozygousdeletions on 9p that do not extend into the CDKN2 locus (Cheng et al.,1994; Lydiatt et al., 1994). In fact, these two reports suggest that oneof the other 9p TSGs is telomeric to CDKN2. To date, all the dataavailable so far in primary tumors and tumor cell lines, suggest thatthe preferred mechanism for gene inactivation on 9p is homozygousdeletion rather than point mutations. No other chromosomal region isknown with such a high frequency of homozygous deletions. It is ratherintriguing that all of the genes (the IFN gene cluster, MTAP, CDKN2 andCDKN2B) identified thus far in this region could have some significantbiological role in cancer. The most efficient way to inactivate all ofthese genes if they are biologically important would be by a largeenough deletion. Alternately, these genes may have been deleted as“innocent bystanders” because intrinsic fragility or recombinogenicityaround the TSG may make the region a hot spot for illegitimaterecombination.

The inclusion of MTAP gene in these deletions may present an opportunityto use this phenomenon in drug development (Carerra et al., 1984; DellaRagione et al., 1992; Nobori et al., 1993). MTAP is involved in thepurine salvage pathway in which methylthioadenosine is recycled to thepurine nucleotide pool. MTAP deficiency interferes with this salvagepathway. MTAP deficiency in human malignancy may permit the developmentof chemotherapeutic approaches in which MTAP-negative cancer cells willbe selectively killed with drugs causing the depletion of purinenucleotides. This major difference between normal and malignant cellsmay be used to design more effective chemotherapy approaches in gliomas,lung cancer and other solid tumors where there are currently noeffective therapies.

5.2 Example 2 Mapping of Genomic Rearrangements Involving the Short Armof Chromosome 9 in ALL and other Hematologic Malignancies

5.2.1 Material and Methods

5.2.1.1 Patients

The karyotypes of patients with hematological malignancies referred tothe Cytogenetic Hematology/Oncology Laboratory of the University ofChicago Medical Center between 1989 and 1994 were reviewed. Twenty caseswith cytogenetic rearrangement of the short arm of chromosome 9 in atleast 30% of the metaphase cells analyzed and for which material wereavailable were selected. Cytogenetically, 7 cases had a rearrangementthat involved no loss of band 9p21, 9 cases retained one copy and 4cases had homozygous deletions of chromosomal band 9p21. Each case (13ALL, 2 AML, 5 NHL) was classified according to the criteria of theFrench-American-British (FAB) classification for leukemias or theworking formulation for the non-Hodgkin's lymphomas (Non Hodgkin'sLymphoma Pathologic Classification Project, 1982; Bennett et al., 1976).Table 3 shows the clinical characteristics of the study patients.

TABLE 3 Characterization of Patient Material % of Copies Abnormal CaseNo. Sex/Age (yr) DX Stage Chromosomal Abnormalities of 9p of 9p21* CloneSample 1 M/33 B-ALL DX t(2; 9)(p12; p23) 2 83 BM 2 M/28 B-ALL DX dic(9;12)(p1?3; p1?2) 1 15 BM dic(9; 12)(p1?3; p1?2), del(9)9p13p22) 0 36 3M/36 B-ALL RL t(3; 9)(q29; p13) 2 100 PB 4 F/66 B-ALL RL der(9)t(8;9)(q11; p13) 1 40 BM 5 M/67 B-ALL DX der(9)t(4; 9)(p1?4; p2?1) 1 100 BM6 F/14 B-ALL RL der(9)t(9; ?9)(p1?2; q22) 1 82 BM 7 M/26 B-ALL DXder(9)t(9; 17)(p13; q11) 1 49 BM 8 F/2 T-ALL DX del(9)(p2?1p24) 1 83 BM9 F/50 B-ALL DX dic(9; 19)(p11; p13) 1 90 BM 10 M/28 B-ALL RLadd(9)(p24) 2 84 BM 11 F/24 B-ALL DX −9 1 40 BM −9, del(17)t(9; 17)(p13;p11) 2 51 12 M/17 B-ALL DX dic(7; 9)(p1?3; p1?1), t(9; 11)(p13; p11) 1100 BM 13 F/17 B-ALL DX del(9)(p13p23) 1 63 BM 14 F/85 AML DX −9,add(9)(p13q34) 0 100 PB 15 M/69 AML DX dic(5; 9)(p15; p13), dic (9; ?;16; ?) 0 100 BM (9qter→9p13::?::16p11→16q22::?), +del(9)t(9; 19)(p11;q11) 16 F/42 NHL, dl DX −9, −9, dic(2; 9)(p2?3; p2?4), 1 73 TUder(9)(p11; q13)† 17 M/64 NHL, fl DX der(9)t(4; 9)(q21;p22)del(4)(q31q33) 2 80 LN 18 M/59 NHL, dl DX t(9; 19)(p13; q13.3) 2 30LN 19 M/69 NHL, dl DX add(9)(p13or21), der(9)t(5; 9)(q11; p12) 0 85 LN20 M/48 NHL, fl DX der(9)t(1; 9)(q21; p23)x2† 4 50 LN Abbreviations: DX,diagnosis; RL, relapse; dl, diffuse large-cell subtype; fl, follicularlarge-cell subtype; BM, bone marrow; PB, peripheral blood; TU, tumor;LN, lymph node. *Number of copies of chromosomal band 9p21 based oncytogenetic analysis. †Near tetraploid karyotype.5.2.1.2 Southern Blot Analysis

High molecular weight DNA was isolated as previously described (James etal., 1988). DNA was digested with the restriction enzyme HindIII,electrophoresed on a 1% agarose gel, and transferred to a nylon-basednitrocellulose membrane (Gene Screen plus, NEN, Boston, Mass.). DNAfilters were hybridized with ³²P-labeled probes from 9p21 and exposed toX-ray film. The chromosomal localization of the 9p probes is shown inFIG. 5. Probe M1.4 represents the 3′ untranslated region of MTAPdescribed in Example 1 which is located approx. 100 kb telomeric toCDKN2. D9S966 is 200 kb centromeric to CDKN2 (Bohlander et al., 1994).CDKN2 (cDNA) and CDKN2B (p15) (exon 1) are located within 20 kb of oneanother Kamb et al., 1994). Equal DNA loading was verified by visualinspection of ethidium bromide-stained gels and by controlhybridizations to a transferrin receptor probe located on chromosome 3(Schneider et al., 1984).

5.2.1.3 FISH Analysis

Mononuclear cells of patients samples, normal bone marrow and peripheralblood cells had been grown in short term culture, harvested usingstandard cell culture techniques and stored in fixative for severalyears (Le Beau, 1994). YAC A88E10 (330 kb), later referred to as YAC 11,was obtained by screening the St. Louis library with IFN A1 primers(Henco et al., 1988). The contig of 8 cosmids encompassing a 250-kbregion around CDKN2 (COSp16) was assembled by screening a flow-sortedhuman chromosome 9 library (Lawrence Livermore Laboratories) with probesfrom a YAC contig of the region. A similar cosmid contig probeidentified all homozygous deletions in leukemia derived cell lines(Dreyling et al., 1994).

YAC 284D6 (320 kb), later referred to as YAC 10/2, from chromosomal band8q22 was used as a control probe (Erickson et al., 1992). pHuR98, avariant satellite 3 sequence from the centromere of chromosome 9, wasused to determine the copy number of this chromosome (Moyzis et al.,1987). FISH probes were prepared using sequence-independentamplification (SIA) as previously described (Bohlander et al., 1992;1994). The copy number of chromosome 8 was determined by a centromericFISH probe CEP 8 Spectrum Orange (Imagenetics, Framingham, Mass.).Two-color FISH with a YAC or cosmid probe and a centromeric probe wasperformed as previously described (Rowley et al., 1990). Briefly, thehybridization solution contained approx. 0.1 mg of each probe, 1 μghuman Cot1-DNA (BRL), 0.6 μg human placental DNA and 3 μg salmon spermDNA/slide in a 10 μl volume. The biotinylated probes were detected withfluorescein isothiocyanate (FITC)-conjugated avidin. The slides werecounterstained with 4′,6′-diamidino-2-phenylindole dihydrochloride, andwere analyzed using epifluorescence and a single pass filter (ChromaTechnology) to avoid superimposition of the centromeric and the cosmidsignals.

For each case, 250 single, intact interphase cells were analyzed.Separate gray scale images of DAPI-stained cells and fluorescencesignals were captured using a cooled charge-coupled device (CCD) camera(Photometrics, Tucson, Ariz.) and were merged using NIH Image (NIH,Bethesda, Md.) or Adobe Photoshop (Adobe Systems, Mountain View,Calif.).

5.2.1.4 SSCP Analysis

Cases with hemizygous deletions of the CDKN2 gene were analyzed by SSCP.HL60, a myeloid cell line with known point mutation in exon 2(nucleotide 232: C→T), was included as a positive control (Nakamake etal., 1994). CDKN2 exons 1 and 2 were radiolabeled with ³²P-dCTP by PCR™using primers 2F and 1108F or 42F and 551R as described previously (Kambet al., 1994). The amplification product was digested with SacII andApaLI, respectively, denatured and run on a 40% polyacrylamide gel under3 different conditions (room temperature, with 10% Glycerol, and at 4°C.).

5.2.2 Results

5.2.2.1 Southern Blot Analysis

In 15 cases DNA was available for Southern blot analysis. Homozygousdeletions on 9p were detected in 8 cases (5 ALL, 1 AML, 2 NHL). M1.4 wasdeleted in 6 cases (40%), CDKN2 and CDKN2B in 8 cases (53%), and D9S966in 7 cases (47%) (Table 4, FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, and FIG.6E). Patient 3 had the smallest deletion which included CDKN2 andCDKN2B, but did not involve the flanking molecular markers. Incomparison, cytogenetic analysis detected homozygous deletions of9p21–22 in only two cases (Patent No. 15 and Patent No. 19), loss of oneallele in five cases, and no loss of 9p21 in one of the 8 cases withhomozygous 9p deletions detected by Southern blot. Thus, in the majorityof cases the deletion was submicroscopic in one of the chromosome 9homologues.

One case showed a significant reduction in the intensity of the Southernblot bands (Patient No. 2). Only 51% of the metaphase cells from thebone marrow had a clonal abnormality of 9p. Thus, the Southern blot datalikely represents a homozygous CDKN2 deletion in a subpopulation ofcells. However, the Southern blot data is also compatible with ahemizygous deletion in the vast majority of the malignant cells.Unfortunately, there was no material available for FISH analysis.

TABLE 4 Southern Blot, FISH, and SSCP Analysis of HematologicMalignancies {PRIVATE} Case Cytogenetic YAC11 COSp16 SSCP No. DXAnalysis M 1.4 CDKN2 CDKN2B D9S966 (FISH) (FISH) CDKN2 1 ALL + + + + + +H N 2 ALL  −†  +*  +*  +*  +* ND ND ND 3 ALL + + − − H ND ND ND 4 ALLH + + + + H H N 5 ALL H − − − − − − ND 6 ALL H + + + + H H N 7 ALL H NDND ND ND H H ND 8 ALL H ND ND ND ND H − ND 9 ALL H + + + + H H N 10ALL + ND ND ND ND H H N 11 ALL  +† − − − − ND ND ND 12 ALL H − − − − − −ND 13 ALL H + − − − ND ND ND 14 AML − ND ND ND ND H − ND 15 AML − − − −− H − ND 16 NHL H − − − − ND ND ND 17 NHL + + + + + + + ND 18 NHL + NDND ND ND + + ND 19 NHL − − − − − H − ND 20 NHL + + + + + + + NDAbbreviations: Dx, diagnosis; +, no deletion; −, homozygous deletion; H,hemizygous deletion; +*, Southern blot bands of reduced intensity; N,normal (no point mutation); ND, not done. †Analysis of largest subclone.5.2.2.2 Interphase FISH Analysis

To determine the reliability of the FISH probes used in this study tentest hybridizations of peripheral blood cells and five hybridizations ofbone marrow cells from normal individuals were performed with eachprobe. Both cosmid contig and YAC probes showed an almost identicaldistribution of signals/cell comparable to previously published resultsfor centromeric probes. In 500 nuclei scored, 2 signals were detected in94–97% of the cells.

Interphase FISH analysis was performed on 15 patient samples. Homozygousdeletions of COSp16 were detected in 6 cases (3 ALL, 2 AML, 1 NHL), fourof whom also showed homozygous deletions on Southern blot analysis(Table 4). Two of the patients had material for FISH studies but not formolecular studies. Cytogenetic analysis detected homozygous deletions of9p21–22 in three cases and loss of only one allele in three of the 6cases with homozygous 9p deletions detected by FISH (Table 4). However,in all samples, the percentage of cells with 9p deletions detected byFISH correlated closely with the percentage of metaphase cells with 9prearrangements detected by cytogenetic analysis. In the cases withhomozygous 9p deletion, 87.7±7.3% of the cells showed no COSp16 signal,while 94.3±2.7% of the cells retained all copies of the control YAC onchromosome 8 (FIG. 7A). Interphase FISH with COSp16 identified allhomozygous deletions detected by molecular analysis. However, four caseswith homozygous deletion detected by Southern blot had no materialavailable for FISH studies. Both copies of the IFN region (YAC 11) weredeleted in 2 ALL cases.

Hemizygous deletions of the CDKN2/CDKN2B region were detected byinterphase FISH in 6 ALL cases (FIG. 7B). Cytogenetic analysis hadpredicted a loss of one copy of chromosomal band 9p21 in 4 of thesecases and no loss of 9p21 in 2 cases (Table 4). These hemizygousdeletions could not be detected by Southern blot analysis, probablybecause only subpopulations of cells (43–86% of interphase cells) wereinvolved. In addition, molecular analysis did not detect any homozygousdeletion of other markers of the region (M1.4 or D9S966) in these cases.

Combined interphase FISH and Southern blot analysis detected CDKN2deletions in all 13 ALL cases (100%; 7 homozygous, 6 hemizygousdeletions), both cases of AML (100%; homozygous deletions) and in 2 of 5NHL cases (40%; homozygous deletions). The two deletions in NHL wereobserved in lymphomas of the diffuse large cell subtype. Thus, 17 of 20cases (85%) had genomic 9p aberrations which included CDKN2. This higherdeletion rate when compared to the cytogenetic analysis (65%; 4homozygous and 9 hemizygous deletions) reflects the detection ofsubmicroscopic deletions by the combined techniques of interphase FISHand Southern blot analysis.

5.2.2.3 SSCP Analysis

Five of the 6 ALL cases with hemizygous CDKN2 deletions had DNAavailable for SSCP analysis. HL60, a myeloid cell line with a knownpoint mutation in exon 2 (CDKN2) was used as control. No abnormal SSCPbands were detected in the 5 patient samples, suggesting the absence ofpoint mutations in these samples (Table 4, FIG. 8).

5.2.3 Discussion

In the present study of 20 primary hematological malignancies withcytogenetically detected rearrangements of 9p, CDKN2 was included ingenomic alterations in 85% of the cases. This confirms that the CDKN2region is the likely target of 9p deletions in these hematologicalmalignancies because 10 of 17 cases (59%) of deletions detected bymolecular analysis affected an apparently cytogenetically normal band9p21.

In addition to homozygous deletions, hemizygous deletions were detectedin 6 patients or 30% of the samples. Southern blot analysis was not ableto detect these hemizygous deletions because of contamination withnormal cells. Previous studies had reported relatively small percentagesof hemizygous deletions (Hebert et al., 1994; Ogawa et al., 1994;Quesnel et al., 1994).

By SSCP analysis, no alteration of the remaining CDKN2 allele could bedetected in any of the five cases with hemizygous deletion examined.Because exons 1 and 2 represent 98% of the coding region of CDKN2, andsince SSCP detects 70–90% of point mutations, the data make thepossibility of point mutations in the coding region of CDKN2 in thesecases very unlikely (Sheffield et al., 1993; Kamb et al., 1994).However, there is a possibility that mutations may be found in thepromoter region or other important regulatory regions in the noncodingpart of CDKN2. Alternatively, the loss of one allele with 50% reductionin the gene dosage may be sufficient for a growth advantage of themalignant cell. This hypothesis remains to be proven. However, neitherscenario explains the predominance of large homozygous deletions in theseries. The absence of point mutations of CDKN2 in the cases withhemizygous 9p deletions may suggest that CDKN2 is not the criticaltarget of 9p deletions in these cases. In other primary tumors such asglioblastoma, point mutations of CDKN2 are also rare (Cairns et al.,1994; Giani and Finocchairo, 1994; Jen et al., 1994; Okamoto et al.,1994). The inactivation of either CDKN2B, which was included in all the9p deletions, or other genes of the region may be important for themalignant phenotype as well. However, it was found that CDKN2B is notdeleted in 4 leukemic cell lines with CDKN2 deletion (Dreyling et al.,1994). The smallest commonly deleted region that could be defined inleukemia derived cell lines and primary tumors is bounded on thetelomeric side by the 3′ end of MTAP and on the centromeric side byCDKN2B. This 120-kb region is included in COSp16; whether additionalgenes can be identified remains to be determined. Moreover, the frequentcodeletion of MTAP, an enzyme involved in the salvage pathway of thepurine metabolism could be of interest to clinicians.

5.3 Example 3 Detection of CDKN2 Deletions in Tumor Cell Lines andPrimary Glioma by Interphase FISH

This example demonstrates interphase FISH analysis on 10 tumor-derivedcell lines (4 glioma, 2 melanoma, 2 non-small cell lung cancer, 2bladder cancer) with rearrangement of the short arm of chromosome 9detected by molecular or cytogenetic analysis and 9 primaryglioblastoma, to determine the accuracy of different probes in detecting9p deletions in tumor cell lines and primary tumor tissue. The exampleillustrates the utility of interphase FISH to detect deletions of theCDKN2 region in primary tumors.

5.3.1 Materials and Methods

5.3.1.1 Cell Lines

10 cell lines were used (4 glioma, 2 melanoma, 2 non-small cell lungcarcinoma, and 2 bladder cancer) that had been well characterized byconventional cytogenetic analysis. The cell lines were obtained from theAmerican Type Culture Collection or from the investigators who hadestablished them. Cytogenetic deletions of the short arm of chromosome 9were detected in 4 of 10 cell lines. The cell lines as well asphytohemagglutinin-stimulated normal peripheral blood cells wereharvested using standard cell culture techniques. Metaphase chromosomeswere prepared as described previously (Le Beau, 1994).

5.3.1.2 Patient Materials

Tumor specimens obtained from 9 patients undergoing biopsy or resectionof brain tumors were frozen in liquid nitrogen and stored at −70° C. Thetumors were graded as glioblastoma multiforme according to the WHOclassification system. Touch preparations were made by touching afreshly cut and thawed tumor surface on a slide. The slides were fixedin methanol:glacial acetic acid (3:1), treated with 5 μg/ml proteinase K(Boehringer Mannheim, Mannheim, Germany), and postfixed in 0.5%paraformaldehyde (Sigma Chemical Co., St. Louis, Mo.).

5.3.1.3 FISH Probes

YAC A88E10 (330 kb), later referred to as YAC 11, and YAC 802B11 (1450kb), later referred to as YAC 23, were obtained by screening the St.Louis and the CEPH YAC libraries with IFN A1 primers (Henco et al.,1988). YAC 883G5 (1100 kb), later referred to as YAC 17, were obtainedfrom the CEPH Mega YAC library by screening with D9S966 primers(Bohlander et al., 1995). YAC 284D6 (320 kilobases), later referred toas YAC 10/2, from chromosomal band 8q22 was used as a control probe(Erickson et al., 1992).

Eight cosmids encompassing a 250-kilobase region around CDKN2 were used.The cosmid contig was assembled by screening a flow-sorted humanchromosome 9 library (Lawrence Livermore Laboratories) with probes froma YAC contig of the region. The exact localization of the FISH probes isshown in FIG. 9. FISH probes were prepared as described previously(Bohlander et al., 1994). YACs were purified on a pulsed-field gel. TheDNA of the excised YAC bands as well as the cosmid DNA (20–100 pg) wasamplified using a SIA (Bohlander et al., 1992). The amplificationproducts were PCR™ labeled with biotin-11-dUTP (Enzo Diagnostics) andfinally treated with DNase (DNase I, 200 pg/ml for 10–20 min: BoehringerMannheim) to reduce the average fragment size to 150–450 bp. pHuR98, avariant satellite 3 sequence, which hybridizes specifically to theheterochromatic region of chromosome 9 (9qh), was used to determine thecopy number of chromosome 9 (Moyzis et al., 1987). The plasmid with a158-base pair insert was amplified by SIA, PCR™ labeled with SpectrumOrange-11-dUTP (Imagenetics, Framingham, Mass.), and treated with DNaseas described. The copy number of chromosome 8 was determined by acentromeric FISH probe CEP 8 Spectrum Orange (Imagenetics).

5.3.1.4 FISH Procedure

Two color FISH with YAC or cosmid probes and a centromeric probe wasperformed as described previously (Rowley et al., 1990). Thehybridization solution contained approximately 0.1 μg of each probe, 1μg of human Cot1-DNA (BRL), 0.6 μg of human placental DNA, and 3 μg ofsalmon sperm DNA/slide in a 10-μl volume. The biotinylated probes weredetected with FITC-conjugated avidin. The slides were counterstainedwith 4′,6′-diamidino-2-phynylindole dihydrochloride and were analyzedusing epifluorescence and a single-pass filter (Chroma Technology) toavoid superimposition of the centromeric and the YAC signals. Forinterphase analysis of the cell lines, the FISH signals of a total of500 single, intact cells were counted by 2 independent observers. Inaddition, 25 metaphase cells of each cell line were analyzed. In thetumor samples 100 single intact cells were analyzed. For FIG. 9,separate gray scale images of 4N,6N-diamidino-2-phenylindole-stainedcells and fluorescence signals were captured using a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and werepseudocolored and merged using NIH Image or Adobe Photoshop.

5.3.1.5 Molecular Analysis

Cell line DNA was extracted and treated with restriction enzyme(HindIII), electrophoresed on a 1% agarose gel, and transferred to anylon-based nitrocellulose membrane (Gene Screen Plus; NEN, Boston). DNAfilters were hybridized with ³²P-labeled probes from 9p21 and exposed toX-ray film. The probes used were REY24, CDKN2 cDNA, D9S966, and D9S171(Bohlander et al., 1995). The exact order of the molecular markers isshown in FIG. 9.

5.3.2 Results

5.3.2.1 Interphase Analysis in Normal Peripheral Blood

To determine the reliability of the FISH probes in nonmalignant cells,ten test hybridizations of peripheral blood cells from normalindividuals were performed with each probe (Table 5). Both centromericand YAC probes showed an almost identical distribution of signals/cellcomparable to previously published results for centromeric probes. In500 nuclei scored, 2 signals were detected in 94–97% of the cells.

5.3.2.2 Interphase Analysis in Tumor Cell Lines

9p deletions were determined by molecular analysis in 9 of 10 cell lines(Table 6). All deletions were detected as well by Interphase FISH withthe COSp16 probe. The results of the FISH analysis are summarized as adeletion map (FIG. 9). YAC 23 was homozygously deleted in one cell line(H322). YAC 11 which covers the proximal IFN gene cluster was absent in2 cell lines (U410, H322); only one copy was retained in one cell line(H4). Moreover, the intensity of the hybridization signals for YAC 11was significantly reduced in 3 cell lines (H290, H4, A172). Previousdetailed molecular analysis revealed that the distal deletionbreakpoints of these cell lines lie within the YAC 11 region (Olopade etal., 1992; 1993). Therefore the intensity of the signal is reduced.However, it was still possible to detect signals for YAC 11 in one cellline [H4] even though 90% of the YAC region was deleted. YAC 17 washomozygously deleted in 2 cell lines (H290, RT4), the number of copieswas reduced in 2 other cell lines (A172, H4), and 1 cell line showed apartial deletion of one allele (1410). The cosmid probe which covers theregion of the CDKN2 was homozygously deleted in 8 of 10 cell lines. Inone cell line (T98) the signal was significantly reduced indicating apartial deletion of the region. Southern blot analysis showed ahomozygous deletion of CDKN2 on this cell line, whereas anothermolecular marker of the region was retained. In 5 cell lines (H4, U410,HS294T, RT4, UM-UC3), both control probes, the chromosome 8 centromereprobe and the YAC 10/2 probe, showed a

TABLE 5 Interphase Analysis of Normal Peripheral Blood FISH Region/ No.of hybridization signals/cell (%) probe marker 0 1 2 3 4 >4 pHuR 9qh 0.02 ± 0.06^(a) 3.22 ± 0.54 95.70 ± 0.50 0.78 ± 0.37 0.32 ± 0.32 0 YAC17 D9S966 0.30 ± 0.27 3.90 ± 1.33 94.66 ± 1.63 0.74 ± 0.39 0.40 ± 0.27 0COS p16 CDKN2 0.10 ± 0.19 2.68 ± 1.00 96.02 ± 1.46 0.82 ± 0.62 0.32 ±0.23 0.04 ± 0.13 YAC 11 IFN A 0.06 ± 0.10 2.12 ± 0.8  96.60 ± 1.10 0.80± 0.49 0.42 ± 0.32 0 YAC 23 IFN A 0.04 ± 0.08 1.92 ± 0.33 97.08 ± 0.650.78 ± 0.69 0.12 ± 0.10 0 YAC 10/2 8q22 0.16 ± 0.18 1.94 ± 0.65 97.04 ±0.65 0.62 ± 0.30 0.24 ± 0.16 0 ^(a)Mean ± SD of hybridizationsignals/cell. Dual color FISH was performed with normal peripheral bloodcells of 10 probands.similar distribution of signals/cell indicating the comparablehybridization efficiency of centromeric and YAC probes. However, in the5 remaining cell lines (all with 4 or more copies of chromosome 8) thenumber of chromosome 8 centromere signals/cell differed from the numberof YAC 10/2 signals/cell suggesting the presence of rearrangementsaffecting chromosome 8.

Subpopulations of cells showed an aberrant number of centromere 8 and 9signals reflecting the heterogeneity of the cell lines. Metaphaseanalysis confirmed these subpopulations of cells with a loss or gain ofa chromosome homologue in 5 of 10 cell lines. However, the heterogeneityof the cell lines did not affect the analysis of 9p deletions, becauseall subpopulations of a cell line uniformly had either deleted orretained the tumor suppressor region on 9p. In the latter group thenumber of signals/cell was highly comparable to the centromere 9 data Incontrast, in the cell lines with homozygous deletions 99.5±0.4% (SD) ofthe cells showed no hybridization signal (FIG. 10A). Nonhomozygousdeletions could be detected with a similar accuracy. Thus, there wasgood concordance between the molecular results and the FISH data. Allthe homozygous deletions determined by molecular analysis were detectedby Interphase FISH (Table 6). In addition, cell lines with a partialloss of the IFN gene cluster had a reduced intensity of thehybridization signal of YAC 11.

TABLE 6 Molecular and Cytogenetic Features of Human Tumor Cell Lines No.Cytogenetic Chrom alterations of 9 Cell line Tumor type chromosome 9Ploidy (FISH) IFNA REY24 CDKN2 D9S996 D9S171 A172 Glioma 9p on marker,16 Near triploid 4 −p^(a) − − − + marker chromosomes H4 Glioma del(9p)3X Hypertriploid 3 −p^(a) − − − + T98 Glioma 14–16 markerHyperpentaploid >4 + + − + + chromosomes U410 Glioma ND Near diploid 2 −− − − + HS294T Melanoma Normal Near triploid 3 + − − + + RPMI7951Melanoma ND Heteroploid >4 + + + + + (>2X66X) H290 NSCLC del(9)t(6;9)(p11; p11), Near tetraploid 4 −p^(a) − − − − del(9)(p11), del(9)(p22)H322 NSCLC del(9)(p13), 25 Near tetraploid 4 − − − − + markerchromosomes RT4 Bladder −9, del(9)(p21p22) 3X Hypotetraploid 3 −p^(a) −− − + carcinoma UM-UC3 Bladder +9, del(9)(q12 or Hypertriploid 4 + − −− + carcinoma q34), add(9)(q12) ^(a)+, autoradiographic signalcomparable to the control; −, no signal; p, partial deletion of the IFNAgene cluster (only some of the multiple bands are present); ND, notdone; NSCLC, non-small cell lung carcinoma. The number of chromosome 9copies were determined by FISH analysis of the centromere 9 probe (pHuR98). The presence of the interferon A cluster (IFN A) and of themolecular markers REY24, CDKN2, D9S966, and D9S171 were determined bySouthern blots.5.3.2.3 Interphase Analysis in Tumor Specimens

To determine 9p deletions in primary tumors, 9 brain tumors,pathologically classified as glioblastoma multiforme, were analyzedusing the FISH probes YAC 11, COSp16, and YAC 10/2 for detection of thedeletion of the proximal IFN gene cluster (YAC 11), the CDKN2 region(COSp16), and a control probe (YAC 10/2). Of 9 tumors, 4 tumors (44%)had a deletion of the proximal IFN gene cluster [YAC 11 (FIG. 10B)]. Nocosmid signal for CDKN2 was detectable in 5 tumors. In tumor sample 1(FIG. 10B), the intensity of the hybridization signals of the cosmidcontig was significantly reduced in comparison to the control YACs,indicating a partial deletion of the cosmid contig.

In tumor sample 7 (FIG. 10B), 7% of the cells did not show any YAC 11signal. This tumor had only one copy of chromosome 9. Therefore, thenumber of cells without hybridization efficiency. In aneuploid tumors (6cases, determined by interphase FISH) a subpopulation of cells(13.4±4.8%) had 2 copies of chromosome 9. This cell population was notidentified in cell lines and probably represents the contamination withnormal cells (stromal cells, lymphocytes, etc.).

5.3.3 Discussion

Interphase FISH analysis is a well established method to determinechromosomal aberrations in hematological malignancies and solid tumors.Using the appropriate probes, interphase analysis is able to detectchromosomal aberrations in clinical tumor specimens contaminated withnormal cells and is also able to detect these changes in smallsubpopulations of cells. In this study, the analysis of 10 cell linesderived from gliomas, melanomas, non-small lung cancer, and bladdercancer and 9 primary gliomas is described using interphase FISHanalysis. For these studies, FISH probes were generated from YACs and acosmid contig by a SIA technique described previously (Bohlander et al.,1994). This procedure yields consistent and strong FISH signals forinterphase analysis. In contrast, single cosmids of the 9p region had ahybridization signal of only moderate intensity due to the small insertsize. At present, FISH probes of YACs or similar vectors have beengenerated previously by Alu-PCR™ (Nelson et al., 1989). Thisamplification technique is limited by the number of Alu sequences perclone which varies considerably. Hybridization of YAC probes generatedby Alu-PCR™ to extended chromatin preparations showed incompleterepresentation of the YAC insert (Tocharoentanaphol et al., 1994).Therefore, Alu-PCR™ generated probes may not accurately detect partialdeletions of the hybridization region.

In these studies, the cosmid contig probe identified all homozygousdeletions of the CDKN2 region in 9 of 10 cell lines. Only 3 of thesecell lines had cytogenetically visible deletions of the short arm ofchromosome 9. The deletions were confirmed by molecular analysis of thecell lines. The remaining melanoma cell lines (RPMI7951) had a rathercomplex cytogenetic rearrangement of the short arm of chromosome 9 butdid not show any deletion of the CDKN2 region. Sequencing data of thiscell line did not detect any mutation within the second exon of CDKN2.The majority of previously described point mutations of CDKN2 werelocated in this region (Cairns et al., 1994; Giani and Finocchiaro,1994; Jen et al., 1994).

The proximal IFN gene cluster was deleted in 4 of the 9 primary tumors.Although the small number of tumors does not allow an estimation of theoverall frequency of 9p deletions in glioblastoma, these results areconsistent with previous studies (Olopade et al., 1992). Another studydetected IFN gene deletions in 50% of the high grade glioma (James etal., 1991). The authors proposed a tumor suppressor activity of theproximal IFN gene cluster in glioblastoma (James et al., 1993). However,the data exclude the IFN genes from the critical region of deletioninasmuch as they were deleted in only 4 of 6 tumors with 9p deletions.The CDKN2 region was deleted in 6 of 9 tumors (67%). Other studiesshowed CDKN2 deletions in 17–69% of the tumors (He et al., 1994; Jen etal., 1994; Schmidt et al., 1994). In a series of primary gliomas,Southern blot analysis showed homozygous deletions of CDKN2 in 45% ofthe tumors. However, Southern blot analysis may miss some of the 9pdeletions because of contamination with normal cells. In addition, it iswell known that some tumors are heterogeneous, and 9p deletions may bepresent in only a subpopulations of cells.

These data suggest that the 250-kb region covered by the cosmid contigincludes the target gene of the 9p deletions in primary glioma. CDKN2 islocated in the smallest region of deletion on 9p. However, the frequencyof point mutations detected in primary tumors is rather low (Giani andFinocchiaro, 1994; Jen et al., 1994). Therefore, the simultaneousdeletion of the neighboring genes may be responsible for the selectivegrowth advantage for the malignant cells. Hannon and Beach (1994)proposed that p15 (M7S2, CDKN2^(b)), a transforming growth factorβ-regulated member of the p16 family, also plays an important role incarcinogenesis. p15 maps approximately 20 kilobases centromeric to CDKN2and is included in the cosmid contig (Kamb et al., 1994). It may well bethat the predominant mechanism of 9p rearrangements in primary tumors isthe deletion of a large genomic region which would inactivate both genesin one step. In fact, in cell lines as well as in primary glioblastoma,the vast majority of deletions include both genes (Jen et al., 1994;Kamb et al., 1994; Nobori et al., 1994). Therefore, it appears thathomozygous deletions are the predominant mechanism for inactivating thisregion. Because further mapping data are crucial to determine theclinical significance of these rather large deletions in primary tumors,FISH will play an important role in characterizing the deletion.

Recently, the over-expression of CDK4, the target molecule of p16, wasproposed as an additional mechanism of functional p16 inactivation (Heet al., 1994; Schmidt et al., 1994). Both events would result in adis-inhibition of the cell cycle. However, in a number of cell lines andprimary gliomas the homozygous deletion of CDKN2 was the much morefrequent event (He et al., 1994; Schmidt et al., 1994).

5.4 Example 4 Identification of Tumor Suppressor Genes Involved inLeukemia

A tumor suppressor gene (TSG) involved in acute lymphoblastic leukemia,gliomas and lung cancers is present on 9p, and is likely to be involvedin the more general pathway of oncogenesis since CDKN2 (p16^(INK4)) is acandidate TSG at this locus. The CDKN2 gene is an attractive candidatefor a tumor suppressor gene because loss of its normal function as aninhibitor of CDK4 could lead to uncontrolled cell growth. Inactivationof CDKN2 by homozygous deletion or point mutation has been reported in alarge proportion of cultured cell lines from multiple tumor types. It ismutated to a lesser degree in primary tumors. In addition, germlinepoint mutations has recently been described in familial melanomaDefinitive proof that CDKN2 is the 9p tumor suppressor gene depends onits ability to suppress tumorigenicity when introduced into cell lineswith deletions. However, there is mounting evidence that CDKN2 may notbe the only biologically relevant gene in this locus.

This example relates to methods of reintroducing identified candidategenes into mouse and human cell lines with deletions by minigene or YACtransfection, methods of identifying transcription units in the criticalregion and testing for alterations of such transcripts in glioma celllines and primary tumors, methods for obtaining full-length clones ofrelevant transcripts from cDNA libraries and characterizing the DNAsequence, structural features, and predicted protein characteristics,methods for examining additional breakpoint junctions in glioma celllines to search for possible clues as to the mechanism of the deletions,methods for determining the clinical significance of 9p loss in gliomas.

5.4.1 Deletion Mapping

Initial observation of deletions or rearrangements of IFNA and IFNB1genes in solid tumors have been extended to other cell types,specifically in glioma derived cell lines, primary glioma samples, andlung cancer cell lines. A number of the cell lines also lack MTAP enzymeactivity (Borrensen et al., 1990). In a few cases, the deletions thatinclude both the IFN gene cluster and the MTAP gene are submicroscopicand interstitial in nature which suggests that these genes or a genevery closely linked to them are the relevant genes. It was possible todetermine the order of the genes on 9p:telomere-IFNB1-IFNA/IFNW-MTAP-D9S126-D9S3-centromere. An SRO was definedfor the deletions to be between the IFN gene cluster and MTAP. Inaddition, a long range map of the IFN gene cluster was constructed whichidentified the 26 IFN genes and pseudogenes on this map. Scaffoldattachment regions (SARs) were also localized in relation to the IFNgenes on this map. Several cell lines have been identified withtelomeric deletion breakpoints within the IFN gene cluster. Several ofthese breakpoints were mapped precisely on this genetic map whichpermitted the cloning of the breakpoint junction in two glioma celllines A172 and A1235. The deletion in the glioma cell line A1235 is partof a complex rearrangement that also involves an inversion while thedeletion in the glioma cell line A172 seems to be a simple interstitialdeletion. The breakpoint analyzed in A1235 represented the distaljunction of this inversion-deletion. That an involvement of “AT” richsequences including SARs and LINE elements was found in the deletionbreakpoints in both cell lines was noteworthy.

5.4.2 Construction of a Long-Range Physical Map

Using the CEPH Mega YAC library, screening was performed with two STSs:One for the IFNA1 gene and the other for the microdissection markerMDS59 (D9S966). The IFNA1 STS flanked the previously defined SRO ofdeletions for the 9p TSG on the telomeric side and the MDS59 STSsflanked the SRO on the centromeric side. Several YACs were found foreach of the two STSs. However, none of these YACs contained both STSs.FISH probes were prepared for each of the positive YACs (about 15 YACs)so that they could be tested for chimerism. Interphase FISH studiessuggested that the two groups of YACs were very close if notoverlapping. Some of the non-chimeric YACs from each group were chosento use for a YAC end rescue protocol. The ends of several YACs weresuccessfully cloned and a physical map of 2.8 Mb was constructed (seeExample 1).

After establishing that the Mega YAC groups from the IFN gene clusterand the microdissection clone MDS59 (D9S966) overlapped, the YAC contigwas converted into a cosmid contig. Hybridization probes were preparedfrom selected YACs and screened a gridded flow sorted chromosome 9cosmid library (Lawrence Livermore Lab; gridded at Oncor) directly withthese probes. Identified were several hundred cosmid clones that weredetected by the YAC probes. After using an IFN consensus probe and oneof the MDS59 YACs for a negative selection screen, 128 cosmids wereidentified that were mainly located between the IFN type I gene clusterand MDS59 thus covering the region of the TSG. These 128 cosmids weretransferred to microtiter well plates and replica filters were preparedwith all of the 128 in a 12×11 array. These filters were probed with allof the overlapping YACs from the region.

More detailed ordering of the cosmids was performed for the region ofthe TSG. This was accomplished by using groups of cosmids that werepositive for a YAC end probe as hybridization probes to put back on thecosmid array. In this way cosmids that overlapped with the cosmids fromthe group could be detected. This approach was very successful andallowed the construction of a cosmid contig of the TSG region.

The cosmids from this ordered contig were then used for severaldifferent strategies to detect expressed sequences: direct screening ofcDNA libraries, exon trapping, and a cDNA selection protocol based onthe capture of sequence independently amplified cosmid fragments bybiotinylated cDNA.

5.4.3 Characterization of Microcell Hybrids

A system has been described which forces stable, non-random retention ofhuman chromosome 9 in somatic cell hybrids and microcell hybrids grownin the medium (Broeker et al., 1993). MTA accumulates in allproliferating cells and, after its breakdown by MTAP, is recycled to thepurine salvage pathway, and to the synthesis of methionine (one of theprecursors of the polyamine biosynthetic pathway). The breakdown of MTAprovides the main source of purines for the salvage pathway (Hall etal., 1990). Therefore cells that lack MTAP die in the presence ofinhibitors of de novo purine synthesis (Borrensen et al., 1990).However, cells deficient in MTAP can be rescued from purine starvationby the introduction of a normal human chromosome 9 after cellhybridization or microcell fusion. This allows the design of a cellculture system that selects for the retention of a normal humanchromosome 9 in somatic cell hybrids.

It has been shown that Mouse L cells and a number of human tumor cellslack MTAP activity and can be killed by adding azaserine (an inhibitorof de novo purine synthesis) to the growth medium (Deville et al.,1991). MTAP competent cells will stop proliferating in the presence ofazaserine but can be rescued by the addition of MTA to the growthmedium. To investigate the hypothesis that a tumor suppressor gene isdeleted in Hs294T (a melanoma cell line that has no deletion of the IFNgenes but lacks MTAP activity), a normal copy of human chromosome 9 wasintroduced into the cells by microcell mediated chromosome transfer(Tompson et al., 1992). In a complimentary study two different copies ofchromosome 9, which contained small deletions on the short arm(9p21–p22) that do not include MTAP were also introduced into Hs294Tcells.

Several colonies of microcell hybrids that contained a normal chromosome9 formed within 4 weeks and attained colony sizes with the equivalent of5–6 population doublings. The (+9) hybrids remained viable on the platesfor an indefinite period of time after cessation of proliferation. Thephenotype of these hybrids is characteristic of the senescence ofdiploid human fibroblast. In contrast, the microcell hybrids thatcontained a chromosome 9 with a small deletion at 9p21–p22 continued togrow for many population doublings and did not senesce. Similar studieswere performed with U87MG, a glioma cell line with a large deletionincluding the IFN genes. The (+9) hybrids continued proliferating forseveral months in 10% horse serum and did not senesce. However, whenexpanded the U87 (+9) hybrids did not produce tumors when injected intonude mice. These results suggest that a gene present on 9p21–p22functions in a pathway that leads to senescence in the melanoma cellline and suppresses tumorigenicity in the glioma cell line. Thus,deletion of DNA sequences in this region would inactivate the locus forsenescence, leading to immortalization of the cell.

This provides the cell with a growth advantage that may contribute toneoplastic transformation in vivo and in vitro. Genomic fragments havebeen obtained from within this SRO which detects the overlappinghomozygous deletions in Hs294T, U87MG and the T98G donor chromosome.This region contains CDKN2. It is possible that the reintroduction ofCDKN2 on the normal chromosome 9p into the Hs294T and U87MG isresponsible for both the cell cycle arrest, and the loss oftumorigenicity. Concordant with this hypothesis, the CDKN2 gene ispresent in the donor hybrids containing a normal chromosome 9p but isabsent from the T98G donor cell line and from the recipient cell lines.It remains to be proved if these effects are mediated by the CDKN2 geneor a closely linked gene or genes.

TABLE 7 Tumorigenicity of Microcell Hybrids Containing a Normal orDeleted Human Chromosome 9 Type of donor # of cells/ #tumors/ Cell Lineschromosome 9 injection # sites % Tumors K562 (leuk) none   5 × 10⁶ 9/9100 KTm3 T98G   5 × 10⁶ 8/8 100 KFm1 ALL patient   5 × 10⁶ 6/6 100 CEM(leuk) none  10 × 10⁶ 10/10 100 CJm3 fibroblast  10 × 10⁶ 0/8 0 CJm4fibroblast  10 × 10⁶ 0/8 0 U87MG (gli) none 2.5 × 10⁶ 10/10 100 UNm20afibroblast 2.5 × 10⁶ 0/8 0 UTm10b T98G 2.5 × 10⁶ 8/8 100 UFm4c ALLpatient 2.5 × 10⁶ 4/4 100 UFm5a ALL patient 2.5 × 10⁶ 8/8 100Hs294T(mel) none 2.5 × 10⁶ 10/10 100

Defining the commonly deleted region in the cell lines used haspermitted obtaining expressed sequences from within this region becauseproof that a TSG9 is indeed present on 9p depends upon introducing thegene into deficient cell lines. Because one could not exclude thepossible involvement of the IFN genes in tumor suppression, U87MG gliomacell line has been transfected with IFNA gene. Results suggest thatconstitutive expression of the IFNA gene in this cell line did notprevent tumor formation in nude mice, though the cells had a slowerproliferation rate.

TABLE 8 Tumorigenicity of Human Tumor Cell Lines and Derived TransducedCells Containing the Human IFNA1 Gene Cell Line # tumors/# sites %tumorigenicity K562 (leukemia) 7/8 87.5 K562pA317a⁹⁻⁴ 0/8 0K562pA317a⁹⁻⁶ 0/8 0 U87MG (glioma) 10/10 100 U87MGpBK89D 8/8 100U87MGpBK89pool 8/8 100 U87MGpSV2Neo 4/4 1005.4.4 Identifying Expressed Sequences in the SRO

The region involved in gliomas has been narrowed to only a few hundredkilobases (about 400 kb) and a cosmid contig has been assembled.Therefore, any gene found in this region is likely to be a candidateTSG9. Two un-methylated CpG islands have been identified in this regionwhich are known to be frequently located close to the 5′ end ofhousekeeping genes. Unique DNA sequences have been isolated rom withinthe cosmids and analyzed them for interspecies conservation, byhybridization to zoo-blots.

By adaptation of a technique that selects coding regions from largegenomic segments such as YACs and cosmids (Call et al., 1990), cDNAinserts have been amplified by the polymerase chain reaction (PCR™)containing a biotinylated nucleotide at the 5′ end. The cDNA is attachedto strepavidin magnetic beads which bind the biotin molecule with adisassociation constant of 10⁻¹⁵. The genomic YAC or cosmid DNA isprepared by undergoing sequence-independent amplification (SIA) toattach specific primers to the DNA. The SIA fragments are hybridized tothe cDNA, washed, then bound fragments are eluted and amplified by PCR™.After amplification, the mixture is enriched for those sequences whichare homologous to coding regions from the cDNA.

One group of cosmids that were used for the selection are located at thetelomeric end of YAC 942A3, spanning approximately 80 kb. Two of theselected fragments were used to identify homologous cDNA clones in abrain cDNA library. Both the selected fragments and the correspondingcDNAs were analyzed by Southern blot. One cDNA (Le19-1) was representedonce in 30,000 clones, while the second cDNA (LE19-2) was representedonly once in 600,000 clones, both in an adult brain cDNA library. Bothclones were mapped by FISH and/or Southern blot to the region on 9p22.Sequence information yielded no significant homology of either cDNA toany other gene in Genbank. Both cDNAs are located 40–60 kb from CDKN2,and either one or both are included in almost every deletion where CDKN2was deleted in cell lines. This indicated that there are other candidatetumor suppressor genes in the region based on the deletion mapping.

A second group of cosmids used in the selection are located between thecentromeric end of Yac 886F9 and the telomeric end of Yac 19 (942A3),which encompasses approximately 150 kb. Twenty-five selected fragments,most of which are unique representatives in the selection, have beenanalyzed. One of the fragments has been used to isolate cDNAs from aforeskin fibroblast cDNA library. The selected fragment has beensequenced, and shows significant homology to an anonymous, expressedsequence in fetal brain, and to a gene located in the MX1 region ofchromosome 21. The fragment maps within the 150-kb region between Yac886F9 and Yac 942A3.

5.4.5 Experimental Methods

The introduction of TSG9 into glioma cells after YAC transfection orgene transfection may be documented by FISH analysis, as well as RFLPanalysis for additional DNA, using polymorphic probes from 9p22. Severalfeatures of the malignant phenotype are assessed in the transfectants.The morphology of parental cells and the “TSG9” transfectants andtransfectant with “irrelevant YAC” or vector alone is examined in greatdetail to determine if there are any differences in their morphology. Invitro growth properties such as serum independence, contact inhibition,saturation density, population doubling time, as well as ability to formcolonies in soft agar may be studied. Breast cancer cell lines and theYAC transfectants may be examined for growth in athymic nude mice byinjecting cells (5×10^(6–1×10) ⁷) subcutaneously into these mice. ForU87MG transfectants, the mice must be supplemented with estrogen sincethe cells are estrogen dependent. Tumors may be initially suppressed inthe mice injected with “TSG9” transfectants, but may later grow due toloss of the transferred YAC. These revertants may be examined byresecting the tumors that form, placing the cells back in culture andexposing them to the selective medium. If the introduced YAC had beenlost, the cultures will revert to sensitivity.

To clone additional genes, each of the exons trapped and each unique DNAfragment is examined. Initial screening is done by hybridizing thefragment to a panel of deletion cell lines and a multiple tissueNorthern blot. Exons that are non-repetitive and deleted in the celllines are sequenced in order to generate STSs that can be useful forfurther screening. Where transcripts are identified, a cDNA library fromthe tissue with a high level of expression is screened. Because of theubiquitous expression of such genes, adult and fetal brain as well asfibroblast oligo-dT primed and random primed cDNA libraries have beenutilized.

Additional cDNA libraries may be prepared using the RNase H method ofGubler and Hoffman with priming with oligo d(T) or randomoligonucleotide hexamers. cDNA libraries are prepared from mRNA whichhas been completely denatured by methylmercuric hydroxide to releasesecondary structures. After second strand synthesis, low molecularweight (less than 400 bp) cDNAs are removed and the remaining cDNAs areselected and cloned into lambda phage vectors. Resulting recombinantmolecules are packaged using commercially available extracts(Stratagene) and the resulting particles plated using E. coli of anappropriate strain. The pre-made libraries come with full protocolhandbook and appropriate methods will be used for bacterial cultureplating, library plating and tittering and making filter replicas.

5.5 Example 5 Inactivation of CDKN2 (p16^(INK4A)) Tumor Suppressor GeneContributes to Tumor Progression in Low-Grade Lymphoid Malignancies

The natural history of low-grade non-Hodgkin's lymphoma (NHL) ischaracterized by a prolonged indolent phase which is followed byprogression to intermediate- or high grade lymphoma with a dismalprognosis. This clinical progression is often associated with detectablehistologic changes but the genetic alterations involved in thetransformation have not been well characterized. The tumor suppressorlocus on the short arm of chromosome 9, CDKN2, encodes a 16 kDa protein(p16^(INK4A)) which acts as a negative regulator of the cell cyclethrough its interactions with RB and CDK4/CDK6 proteins. To determinewhether CDKN2 is involved in the transformation to diffuse large celllymphoma (DLCL), 11 cases of de novo DLCL; 5 cases of DLCL which evolvedfrom low grade NHL (transformed DLCL); and 9 low grade NHL which hadsubpopulations of large cells with diffuse growth pattern (7 follicularNHL, 1 CLL, 1 mycosis fungoides) were examined. Interphase FISH wasperformed on these samples using a 250-kb cosmid contig (COSp16)encompassing CDKN2. One of 11 cases of de novo DLCL (9%) and 1 of 9 lowgrade NHL (11%) had homozygous deletions of COSp16. In contrast, allfive transformed DLCL (100%) has COSp16 deletions. Two cases hadhomozygous COSp16 deletions, two cases had hemizygous deletions, and onecase had a partial homozygous deletion of the cosmid contig. Thus, CDKN2is frequently deleted in transformed DLCL in contrast to de novo DLCL(p<0.01, Fisher's exact test) or low grade NHL (p<0.03). In addition toits critical role in ALL, these results suggest that CDKN2 deletion is agenetic marker for the histologic transformation from low grade todiffuse large cell lymphoma.

5.6 Example 6 Genomic Cloning and Characterization of the MTAP Gene

This example describes the nucleotide sequence, expression and genomicorganization of the human MTAP gene.

5.6.1 Materials and Methods

5.6.1.1 Screening of cDNA and Genomic Libraries

To obtain YAC end-specific probes, YAC end-rescue reactions were carriedout as described by Hermanson et al., 1991. A YAC end rescued insertsfrom YAC 24 was used as probe to screen the Lawrence Livermorechromosome 9 specific cosmid library. In addition, a cosmid contig ofthe critical region on 9p21 was assembled as described. These cosmidswere used in exon-trapping studies using the Exon Trapping System(Gibco, BRL). Exon 18–11 trapped from cosmid 18 was used to screen aλgt11 human foreskin fibroblast 5′ stretch cDNA library from Clontech. A2.5-kb cDNA was obtained and subcloned into pBluescript SK+ (Stratagene)producing the two clones, pM1.1 and pM1.4. The cosmid contig subarraywas rescreened with probes M1.1 and M1.4 to identify cosmid clonescorresponding to the entire MTAP cDNA.

5.6.1.2 Determination of Exon/Intron Boundaries

The genomic organization was determined by restriction enzyme digestionand southern blot analysis. Mini-DNA preparations from individualbacterial colonies containing recombinant cosmids were performed usingstandard procedures. The cosmids shown in FIG. 11 were used for mappingand sequencing reactions. A Southern blot from each cosmid clonedigested with HindIII was hybridized with probes containing variousparts of the cDNA as shown in FIG. 12. The probes were generated by PCR™amplification of the MTAP cDNA clone using primers designed from theregion. The Southern blots were washed with at high stringency in0.1×SSC and 1% SDS at 65° C. for 45 min. Exon-intron boundaries weresequenced directly from cosmid DNA using either the Sanger dideoxy chaintermination method using a Sequenase™ kit (United States Biochemical) orby cycle sequencing using the ABI PRISM Cycle Sequencing Kit on a 9600Perkin Elmer thermocycler using appropriate oligonucleotides as primers.

The genomic sequences were compared to the cDNA sequence to establishthe exon-intron boundaries. Sequence analysis was performed usingMacVector 4.15KSB1, Pustal Sequence Analysis software (IBI), and theGenBank database using the BLASTN and BLASTP programs. These nucleotidesequences have been submitted to GenBank™.

5.6.1.3 Long Range PCR™

Long range PCR™ was performed to determine the length of introns usingElongase as previously described. PCR™ reactions were carried out in avolume of 50 μl containing 100 ng of genomic cosmid or placental DNA, 1×Elongase buffer (60 mM Tris-SO₄, pH 9.1) at 25° C., 18 mM (NH₄)₂SO₄ and1.7 mM MgSO₄; 200 mM of each dNTP; 200 μm each of forward and reverseprimers, and 1 U of Elongase. A 30 sec, 94° C. elongation step wasperformed followed by 30 cycles of denaturation (94° C. for 30 sec),annealing (58° C. for 30 sec) and extension (68° C. for 5 min).

5.6.1.4 Localization of MTAP Transcription Start Site

The technique of 5′-RACE amplification by PCR™ was performed todetermine the MTAP mRNA transcription start site. A human placental5′-RACE-ready cDNA kit from Clontech was used. The resulting PCR™products were gel purified and submitted to double-stranded DNAsequencing as described above.

Because human multiple tissue northerns (Clontech) probed with pM1.1 andpM1.4 showed two major transcripts in all tissue types, 3′-RACE was alsoperformed to determine if a longer cDNA or an alternate polyadenylationsignal could be obtained.

5.6.2 Results

5.6.2.1 Homology Analysis of the hMTAP Gene

The MTAP gene has been highly conserved through evolution as shown afterhybridization of the cDNA to an evolutionary blot shown in FIG. 13 atlow stringency. The nucleotide sequence of the 2.5-kb cDNA revealed acontinuous open reading frame of 345 amino acids extending fromnucleotide number 10 including the initiator methionine codon. The DNAsequence revealed no homology and protein sequence comparison with thehighest scoring protein sequences showed only a 47% (54 of 114 aminoacids) homology to a hypothetical 25.8-kDa protein in the petC region ofRhodospirillum rubrum. The homology detected with other purinenucleoside phosphorylase (PNP) was lower but extended over a slightlylarger region in eukaryotic proteins, human, mouse and S. cerevisiaePNPs. It is known that MTAP is the counterpart of PNP but both work ondifferent substrates.

5.6.2.2 Exon/Intron Structure

After screening the cosmid array with MTAP cDNA, 5 cosmids (cosmids 18,28, 29, 31, and 81) were identified using the MTAP cDNA probe. A HindIIIrestriction enzyme map was constructed for the MTAP genomic region usingmultiple approaches. The MacVector program was used to perform a HindIIIrestriction enzyme analysis of the pM1.1 and pM1.4 cDNA sequences. Thesizes of the introns were determined after performing long range PCR™ oncloned genomic cosmid DNA.

Intronic sequences were obtained by direct sequencing from the cosmidsusing reverse and forward primers from the 5′ and 3′ ends from differentparts of the cDNA. This permitted the definition of many exon-intronboundaries. MTAP was found to be composed of 7 exons within 22 kb of DNA(FIG. 11). All the HindIII sites identified are shown in FIG. 13.

A HindIII was found within exon 5 at base pair 658 and within exon 6 atbase pair 856 of the pM1.1 cDNA clone. A third HindIII site was foundeight base pairs 5′ of the poly(A) tail in pM1.4. Using sequencingprimers CF and EF (Table 10) on cosmid 31 DNA enabled the location ofHindIII sites thirteen base pairs into intron 3 and three hundred andten base pairs into intron 4.

Southern blot hybridization of HindIII-digested placenta and genomiccosmid DNA using probes generated from the pM1.1 and pM1.4 cDNA clonesrevealed multiple bands as shown in (map 1 and 2). The sizes of eachfragment and the probes used are seen in FIG. 12 and Table 9.

TABLE 9 Methylthioadenosine phosphorylase Exon-Intron OrganizationSequence at Exon-Intron Junction Preceding or Exon Exon IntronInterrupted No. Size (bp) 5′ Splice Donor 3′ Splice Acceptor Size (bp)Amino Acid 1 145 GCCGTGAAGgtgaga tcttagATTGGAATA ~3600 Lys (SEQ ID NO:3)(SEQ ID NO:4) 2 87 TTTGGCAAGgttaat atgcagCCATCTGAT ~1100 Lys (SEQ IDNO:5) (SEQ ID NO:6) 3 59 CTTGCAAGgtatgg ccatagGCATGGA ~1200 Arg (SEQ IDNO:7) (SEQ ID NO:8) 4 271 ACGAGAGAGgtgtgt ttctagGTTCTTATA ~4000 Glu (SEQID NO:9) (SEQ ID NO:1O) 5 240 GAGGAAGCAgtaggt ctctagGTTTCGGTG ~4000 Ala(SEQ ID NO:11) (SEQ ID NO:12) 6 123 AACCTGAAGgtaagt atccagAATATGGCC~2400 Lys (SEQ ID NO:13) (SEQ ID NO:14) 7 1269 TTGCTTTTTtaactc (SEQ IDNO:15) (cleavage site for polyadenylation) 113 bp = 5′ untranslatedregion 1230 bp = 3′ untranslated region

TABLE 10 Portion Portion of Size (kb) of Source of of Exon IntronHindIII Bands DNA that Hy- Probe Cont. in Cont. Detected with bridizedto Hybridized Probe in Probe Probe Probe Probe A 1 2.1 cosmid 55A11probe AF-AR 1 2.3, 2.1, 18 placental probe 1.2 2 2.1 cosmids 55A11 and65G7 probe 3.8 1, 2 1 1.7, 2.3 cosmids 55A11 and 28 65G7 probe BF-BR 22.3, 2.1, 1.8 placental probe CF-CR 3, 4 2.3, 1.8 cosmids 55A11 and 65G7Probe C 4, 5 1.8 cosmids 55A11 and 65G7 Probe C 4, 5 .4 cosmid 225B8probe EF-ER 5 3.0, 1.8, .4 placental probe EF-ER 5 .4 cosmid 225B8 probeEF-ER 5 cosmids 55A11 and 65G7 probe EF-ER 5 .4, 3.0 cosmid 74B1 probeFF-FR 5 .4, 1.4 cosmid 225B8 probe GF-GR 5, 6 1.4, 2.8 cosmids 74B1 and225B8 Probe D 5, 6 1.4, 2.8 cosmids 74B1 and 225B8 Probe E 6, 7 1.7, 2.0cosmid 225B8 Probe E 6, 7 2.0 cosmid 69B12 probe M1.4 7 2.0 placental

To confirm the positions and to rule out additional HindIII sites withinthe introns, long range PCR™ amplification using Elongase (LifeTechnologies) was performed and the resulting amplified genomicfragments were digested with HindIII and analyzed by gelelectrophoresis. Some of the amplified products were hybridized back toHindIII digested genomic cosmid and placenta DNA to confirm thespecificity of the amplification. Cosmid 28 was used as a template forintrons 1, 2, and 3 and cosmid 81 was used as a template for introns 4,5 and 6. Primers AF-BXR generated a 3.8-kb band containing 145 bps fromexon 1, ˜3.6 kb from intron 1, and 87 bps from exon 2. When digestedwith HindIII, ˜1.7 kb, ˜1.2 kb, and ˜0.9 kb bands were produced. Probe Ahybridized to the 0.9 kb fragment and probe BF–BR hybridized to the1.2-kb fragment.

The 1.7 kb band was not seen with hybridization when using the pM1.1cDNA probe however it is seen on cosmid 18 DNA with hybridization of thep3.8 kb probe. The 1.7-kb fragment is a single HindIII fragment made upentirely of intronic DNA. The 0.9-kb fragment hybridizes to the ˜2.1-kbHindIII band of exon 1 and the 1.2-kb fragment hybridizes to the 2.3-kbHindIII band of exon 2. Intron 2 showed no digestion with HindIII. Forintron 3 there was no noticeable digestion of the fragments because theprimers used were very close to the HindIII restriction sites. Primerpair EF-ER produced a ˜2.8-kb fragment that was reduced to ˜2.1-kb,˜0.5-kb and ˜0.25-kb bands when digested with HindIII. When hybridizedback to cosmid DNA the ˜2.8-kb fragment identified an ˜1.8-kb, 2.1-kb,and ˜0.4-kb HindIII bands.

Thus, an ˜2.1-kb HindIII fragment lies within intron 4. The ˜4.2-kbfragment (83 bp from exon 5, ˜4.0-kb from intron 5, and 69 bp from exon6) generated by primer pair GF,GR was reduced to ˜1.35-kb and ˜2.8-kbfragments. The ˜2.6-kb fragment generated by primer pair HF,HR (53 bpfrom exon 6, ˜2.4-kb from intron 6, and 98-bp from exon 7 and 3′untranslated region) was reduced to an ˜1.7-kb and ˜0.9-kb fragmentsThese results confirmed the presence of only one HindIII site withineach of introns 5 and 6. A genomic map displaying the size of theintrons and the relative position of HindIII sites is shown in FIG. 15.

5.7 Example 7 Lack of p16^(INK4A) and/or pRB in Resected NSCLCCorrelates with Locally Advanced Disease

The tumor suppressor gene CDKN2/p16^(INK4A), located on 9p21 isfrequently inactivated in diverse human tumors by homozygous deletionsor de novo methylation of the 5′CpG island. A reciprocal relationshiphas been demonstrated between pRB inactivation and a lack of p16 proteinexpression in human lung cancer cell lines and primary tumors. Theexpression of p16 and pRB in 39 resected primary NSCLC tumors wasexamined by western blot analysis. The results were correlated withclinico-pathologic features such as histology, tumor size, nodalmetastases and pleural invasion. Ten tumors (26%) were p16(−)/pRB(+); 12(31%) were p16(+)/pRB(−); 5 (13%) were p16(−)/pRB(−); 12 (31%) werep16(+)/pRB(+). Thus, 27 (69%) tumors were p16(−) and/or pRB(−). Tumorslacking p16 and/or pRB were significantly larger than p16(+)/pRB(+)tumors (median size 4 vs. 2.75 cm; p=0.02). However, p16 expressionalone did not correlate with any clinico-pathologic features. Thesestudies suggest that the absence of p16 and/or pRB is the most commonabnormality in NSCLC and support a common growth-regulatory mechanismthat is disrupted in the majority of lung cancers (FIG. 16).

5.8 Example 8 Mapping the Chromatin Structure of the Type I InterferonGene Cluster on the Short Arm of Human Chromosome 9

This example describes the construction of a long-range restriction mapof the type I human interferon (IFN) gene cluster which maps to a 450-kbregion on chromosome 9, band p21. The gene family consists of 1 betagene (IFNB1), 13 alpha (INFA) genes, 1 alpha pseudogene (IFNAP), 1 omega(IFNW) gene, 6 IFNW pseudogenes, and 4 unclassified pseudogenes (IFNP).The chromatin structure has been determined for the type I IFN genefamily in a human hematopoetic cell line, using the Li2′,3′-diiodosalicylate (LIS) scaffold attachment region (SAR) mappingassay. Six separate IFNA or IFNW coding sequences were devoid ofattachment sites. By hybridization of the IFNA2 and IFNW1 coding regionsto non-SAR and SAR DNA fractions, 22 strong SARs were mapped to theflanking regions of the 13 IFNA true genes, 3 strong SARs to theflanking regions of 2 IFNW true gene members, and 2 strong SARs flanked1 IFNW pseudogene. Three weak SARs were mapped in the flanking region oftwo IFNW pseudogenes and one weak SAR mapped to the flanking region ofan IFNA true gene. Likewise, one 3′ and 2 5′ strong SARs flanking IFNA2were identified, whereas both IFNP11 and IFNP12 were flanked by weakerSARs. A similar pattern of SAR distribution throughout the type I IFNgene family was observed in a human glioma cell lines, suggesting thatthe structural organization in which SARs define this region into aseries of small DNA loop domains is conserved in different tissue types.

5.8.1 Materials and Methods

5.8.1.1 Cell Lines

For isolation of the DNA scaffold fraction, the chronic myelogenousleukemia (CML) cell line BV173, and the glioma cell line, U373, wereused. BV173 cells are lymphoblastoid precursor B-cells derived from aCML patient in blast crisis. The primary clone in this cell linecontains one normal chromosome 22 and three copies of the rearrangedder. In BV173 the IFN gene cluster on 9p is in a normal germlineconfiguration. U373 cells were derived from a human glioblastomamultiforme tumor. U373 demonstrates functional and biologicalheterogeneity in vitro and in tumors passed in vivo in nude mice.Molecular analysis show no rearrangement at the IFN locus on chromosome9.

5.8.1.2 Cell Cultures

BV173 cells were maintained in RPMI medium supplemented with 10% fetalcalf serum, 1% hepes, sodium bicarbonate (amount adjusted per lot), 1%penicillin, and streptomycin. A fraction of the cells were strained withTrypan Blue to estimate cell viability, and were counted every fourdays. To maintain the line, the cells were plated at 0.5×10⁶ density anddivided 1:4 every 4 days. U373 cells are adherent cells and weremaintained in DMEM and 10% FCS in 175 cm tissue culture flasks.

5.8.1.3 Nuclei and Nuclear Scaffold Isolation—in Vivo SAR Mapping

Methods for isolation of BV173 and U373 cell nuclei and scaffolds havebeen described elsewhere (Stirssel et al., 1995). For these studies,approximately 1×10⁶ cells/ml with 90–100% viability were extracted perstudy. U373 cells were grown to log phase at 80=9% confluency(approximately 5–50×10⁶), placed on ice and washed two times inphosphate buffered saline, then the cells were removed gently from theflask using a rubber policeman.

5.8.1.4 Pellet (Scaffold) and Supernatant (Loop) DNA Purification

To obtain purified pellet and supernatant DNA, each fraction was treatedafter isolation in a standard solution of Tris buffer containing SDS andproteinase K. Scaffold proteins were then extracted with phenol,phenol/chloroform/isoamyl alcohol, and chloroform/isoamyl alcohol. DNAwas ethanol-precipitated and resuspended in sterile water.

5.8.1.5 DNA Clones

The IFNA2 gene was previously mapped and cloned within two overlappinghuman genomic clones (λ 1-1, a 10 kb HindIII fragment, λ 1–2, a 12 kbBamHI fragment). The 10 kb HindIII clone extended further 3′ from thegene than the 12.0 kb BamHI clone. A third λ clone (1-3, a HindIII 11.6kb clone, which overlapped the 5′ end of the BamHI fragment and includedthe IFNP11 gene was also isolated. A fourth 20 kb HindIII λ clone 1-4included IFNP12 and IFNA8 genes. For IFNA5, a 19 kb BamHI λ clone wasmapped, and fragments containing the coding region and the 3′ generegion were isolated and used as probes.

5.8.1.6 Probes and Isolation of Inserts

The technique of Vaux was used to isolate DNA fragments from agarosegels for use as probes on Southern blots. Restriction fragments from λclones containing the IFNA5, IFNA2, IFNP11, IFNP12 coding regions, andtheir 5′ and 3′ flanking regions, were recovered and used as probes inthis study. The IFNW1 coding region was recovered from agarose and usedas a probe. Additional cloned coding regions isolated from plasmids andused for hybridization to supernatant and pellet Southern blots were theIFNW1, IFNB1, the 0.73 kb BamHI exons 5,6,7,9,10 MLL cDNA, the 0.4 kbPstI MLL exons 4,5, and the BCR PstI cDNA 3′ fragment provided by OwenWitte. Additional DNA fragments used as probes on supernatant and pelletgenomic fractions were the IFNB1 1.5 kb EcoRI/PstI 5′ SAR fragment, andthe ph10, ph15 DNA fragments from the BCR gene 1st 90 kb intron.

5.8.1.7 Southern Blot, DNA Hybridization, and Autoradiography

For SAR analysis 4 μg (determined by optical density readings) of thesupernatant and pellet fractions per study were used for Southern blotanalysis. All prehybridizations and hybridizations followed standardmethodology. Autoradiographs of Southern blots were scanned (HP ScanjetII cx) and the label of specific bands was quantitated by MOCHA (Jandel,San Rafael, Calif.) as the mean intensity of the negative imagemultiplied by the band area (number of pixels). The relative strength(R) of a specific DNA segment to partition in the pellet fraction wasestimated as the band intensity in the pellet divided by the sum ofintensities of pellet plus supernatant (R=IP P+IS). Various genefragments were used as positive and negative control fragments to setstandards for binding affinity and were the following: 1) positivecontrol gene fragments showing >70% (R=70) enrichment into the pelletwere the IFNBI 5′ SAR detected with the 1.5 kb EcoRI/PstI probe, and theMLL breakpoint cluster region telomeric SAR detected with the 0.73 kbBamHI cDNA probe; 2) negative control fragments showing negligiblebinding with <30% (R=<30) enrichment into the pellet were the IFNBIcoding region, the MLL breakpoint cluster region non-SAR fragmentsdetected with the 0.73 kb BamHI cDNA probe, and several non-SARfragments from the BCR 3′ gene region detected with the BCR PstI 3′probe; 3) weaker binding control fragments demonstrating 40–60%enrichment into the pellet (R=40–60) were IFNBI 3′ weak SAR detectedwith the IFNBI coding sequences, the MLL weak centromeric SAR detectedwith the 0.4 kb PstI cDNA probe, and the BCR weak intronic SARs mappingwithin the 1st intron detected with probes Ph10, 15. For a particularDNA fragment detected in the supernatant or pellet, these values equalthe total amount of this DNA fragment in each genomic fraction. Thus,this value represents the total pellet or supernatant enrichment forthis DNA fragment in the whole genome.

5.8.2 Results

5.8.2.1 SARs and the IFNA and IFNW Genes in BV173 Hematopoetic Cells

IFNA genes, cut with HindIII and EcoRI were mapped for SARs (FIG. 17A).BglII supernatant and pellet fragments were also analyzed. In addition,HindIII, EcoRI and BglII supernatant and pellet fragments containingIFNW1 coding regions were mapped for SARs. These probes hybridized onlyto genes within their own family as previously described. Using detailedrestriction maps and evaluating the enrichment patterns for all thesegene fragments, SAR localization was obtained (FIG. 17A, FIG. 17B, Table11).

TABLE 11 Gene SAR Location Non-SAR IFN fragments R = ≦30 coding regionsIFNA1 ″ IFNA2 ″ IFNA8 ″ IFNA9 ″ IFNA15 ″ IFNA18 ″ Strong Specific IFNSARs 5′ SAR 3′ SAR IFN21 — 3′ SAR IFNA7 — 3′ SAR IFNA16 5′ SAR 3′ SARIFNA17 5′ SAR 3′ SAR IFNA14 5′ SAR — IFNA5 5′ SAR 3′ SAR IFNA13 5′ SAR —IFNW19 — 3′ SAR Total = 11 SARs IFNA 5′ SARs = 5 IFNA 3′ SARs = 5 IFNW3′ SARs = 1 Strong SARs Mapping both 5′ and 3′ IFNW1 IFNA4 IFNA10 IFNA6IFNA1 IFNA8 IFNA2 IFNPW18 Total = 16 strong SARs weak SARs 5′ SAR 3′ SARIFNA21 5′ SAR IFNPW15 5′ SAR 3′ SAR IFNPW9 3′ SAR total = 4 weak SARs

In Table 11, non-SAR and SAR mapping results are illustrated using theIFNA and IFNW coding regions as probes to supernatant and pellet DNAfractions. Top set of columns show non-SAR DNA fragments detected withthe IFNA2 and IFNW1 coding regions. Middle set of columns demonstrate 8IFN genes and the number and location of specific SARs to the 5′ and 3′flanking gene regions. Note, that the 3′ SAR of IFNA5 (underlined) wasmapped using cloned DNA fragments. All these IFN SARs mapped to DNAfragments containing coding sequences plus 3′ flanking DNA. Bottom setof columns (left) identify strong SARs mapping to both the 5′ and 3′flanking regions of the IFN genes. The bottom (right) column representsthe location of weak SARs to the 5′ and/or 3′ flanking regions of oneIFNA true gene and two IFNPW genes.

Several supernatant enriched fragments (R≦30) were observed containingonly the coding regions or coding regions plus short flanking DNAstretches for 3 IFNA and 3 IFNW genes (Table 11). These resultsdemonstrated that 6 IFN coding regions are scaffold free regions. Thusit is likely that all the IFN coding regions do not represent SARs.Three different groups of fragments were also observed which wereenriched in the pellet fraction (R≧70) (Table 10). The first group offragments, contained coding sequences plus 5′ flanking DNA, the secondgroup contained sequences with only 3′ flanking DNA. These fragmentsallowed the mapping of SAR locations very specifically to either the 5′or 3′ flanking regions. Five strong SARs were mapped to the 3′ flankingregion, and five strong SARs to the 5′ flanking regions of 7 IFNA genes.The 3′ SAR of IFNA5 was mapped using specific DNA fragments from alambda clone. One strong SAR mapped to the 3′ flanking region of oneIFNW gene. The third group of pellet enriched DNA fragments containedcoding regions and approximately equal amounts of both 5′ and 3′flanking DNA partitioning into the pellet fraction (R≧70). It appearedthat high affinity SARs flanked these gene coding sequences. First, sixIFN coding regions were devoid of SARs. Second, in studies where strongSARs could be specifically mapped to the 5′ or 3′ flanking gene regions,three genes contained strong SARs in both flanking regions (Table 11).Finally, one fragment which extended equally both 5′ and 3′ of IFNA2,SARs were specifically mapped to the flanking regions using cloned DNAfragments. Two strong SARs were identified in the third group of pelletenriched DNA fragments: one in the 5′ and one in the 3′ gene regions.Results demonstrated 12 strong SARs to 6 IFNA gene flanking regions, andfour strong SARs to 2 IFNW flanking regions.

Two final groups of DNA fragments were analyzed which contained IFNgenes demonstrating a more equal distribution between the pellet andsupernatant fractions (R=40–60). The first group of DNA fragmentsoverlapped sections of previously identified strong SARs, indicatingthese regions probably contained fragments of strong SAR binding sites.The second group of weaker binding DNA fragments contained separate weakSARs. Weak SARs were mapped to the 5′ flanking region of an IFNA truegene (1 SAR), to the 3′ flanking region of an IFNWP gene (1 SAR), and tothe 3′ and 5′ flanking region of an IFNWP gene (2 SARs).

5.8.2.2 IFNA2 Telomeric and Centromeric SARs in BV173 Cells

Initially observed was a 5.0 kb EcoRI pellet enriched fragment afterhybridizing the IFNA2 coding region to supernatant and pellet fractions.This fragment contained the IFNA2 coding region plus both 5′ and 3′flanking sequences (FIG. 17A). To see if SARs mapped to the geneflanking regions, specific DNA fragments representing the flankingregions of IFNA2 were hybridized to supernatant and pellet fractions.Hybridization of proves a, d (IFNA2 coding region) h, and i tosupernatant and pellet fractions, showed a 1.2-kb HindIII/EcoRI, a0.4-kb BglII, a 1.6-kb EcoRI/BglI and a 1.0-kb BglII/BamHI fragment, allof which enriched to the supernatant fraction and thus defined thelocation of non-SARs (FIG. 17A, FIG. 19A, and FIG. 19B). Hybridizationof probes b, c, f, and g, to various single and double digests showedpellet enrichment for a 4.6-kb EcoRI, a 2.65-kb EcoRI/BglII, a 3.1-kbEcoRI, and a 1.1-kb EcoRI fragment, thus identifying two strong affinitySARs, one 3′ (SAR1) and one 5′ (SAR2) of the IFNA2 gene (FIG. 19A).Hybridization of probes j and k (FIG. 19B) identified an 0.7-kb EcoRIfragment (SAR3) and a 1.8-kb BamHI/EcoRI fragment (SAR3) almostexclusively in the pellet fraction. Probe 1 identified a 3.0-kbEcoRI/BglII fragment which distributed approximately equally betweenboth fractions (FIG. 19A and FIG. 19B).

The region containing the IFNA2 SAR3 and the adjacent weaker bindingregion, were further defined with restriction enzymes which cut morefrequently (4-bp recognition sites) to map the scaffold binding sites.Hybridizing probe 1 to Sau3AI, HaeIII, and EsaI supernatant and pelletfractions, several small supernatant, weak, and pellet enriched DNAfragments were identified (FIG. 20). The SAR3 was separated by 707 bp ofnon-scaffold DNA from a weak affinity SAR (SAR4) (FIG. 20). Oneadditional weak SAR mapped between IFNP11 and IFNP12 (SAR5) (FIG. 19A),and one mapped just 3′ to IFNP12 (SAR6) (FIG. 19A).

5.8.2.3 SARs and the IFNA Gene Cluster in U373 Glioma Cells

To determine if these SARs are conserved in different tissue types, theIFNA SARs were analyzed in U373 glioma cells. As shown in FIG. 20,HindIII and EcoRI supernatant and pellet fragments were analyzed forSARs using the IFNA2 probe. BglII supernatant and pellet fractions werealso analyzed for SARs. A pattern of hybridization similar to that seenwith BV173 cells was observed. These results indicate that SARs areconserved between two different cell types (hematopoetic and gliomacells), in the IFNA2 region. In addition, it was possible to demonstatethat SAR3 and the IFNB1 5′ SAR also showed pellet enrichment similar tothat in BV173 cells.

5.8.2.4 Type 1 IFN SARs

The 450-kb region containing the IFNA and the IFNW genes as shown bydetailed analysis and the 20-kb region containing IFNA2, IFNP11 andIFNP12 is a scaffold rich region (FIG. 16, FIG. 18, and FIG. 19A). Therelative scaffold binding affinity was calculated for 17 different IFNAgene fragments in both BV173 and U373 cells. Enrichment patterns weresimilar for both cell lines. Twenty DNA fragments from the IFNA2, IFNP11and IFNP12 cloned region were measured (FIG. 17A, FIG. 17B, FIG. 18,FIG. 19A, FIG. 19B, and FIG. 21). The strength of scaffold bindingbetween the IFN gene fragments and the different positive and negativecontrol fragments compared well. For example, all IFNA pellet enrichedfragments with R≦30 demonstrating negligible binding compared well withthe IFNB1 coding region (R=3), and non-SAR fragments from the MLLbreakpoint cluster region (R=7) and BCR 3′ gene regions (R=13). None ofthese fragments (IFNA, IFNW or controls) were observed partitioning intothe pellet non-specifically-because of their high molecular weight inmultiple studies. Thus, the results demonstrate specific scaffoldbinding for these gene regions.

In BV173 and U373 cells it was not possible to assign SARs to a fewregions because the restriction sites were not mapped and specific DNAprobes were not available. These gene regions included the 3′ region ofIFNA14 and the 5′ region of IFNWP5 (FIG. 18). Additional regions notmapped were the 5′ region of IFNP23, and 5′ and 3′ regions of IFNAP22,and three 50 kb regions containing no known coding sequences:1) theregion between the IFNB1 gene and the first group of IFNW and IFNA genes2) the region between the first and second group of IFNW, IFNA genes,and 3) the region between the last group of IFNA and IFNW genes and theIFNA1, IFNP23 and IFNWP19 genes (FIG. 18).

5.8.3 Discussion

35 SARs have been mapped within the IFNA and IFNW gene cluster in BV173cells. 23 IFNA SARs have also been shown to map to the same locations inU373 cells. The chromatin organization of the IFN genes has revealedseveral important features including coding regions flanked by SARs,large high affinity SARs demonstrating cooperative interactions and theassociation of weak SARs with some of the pseudogenes. It is probablethat the IFN gene family arose from one primordial gene followed by geneduplication and divergence. The IFNA and IFNW genes have clearly arisenthrough gene duplication and gene conversion events plus an inversion.

At least some pseudogenes are associated with weak SARs. The IFNPW9 geneis probably associated with a 3′ weak SAR and the IFNPW15 is alsoflanked by 5′ and 3′ weak SARs. However, the IFNWP18 gene was flanked bystrong SARs. Since only the IFNPW9 and IFNPW15 pseudogenes were able tobe mapped using the IFNW1 coding region as a probe, it was not possibleto confirm if these weak SARs represent portions of high affinity SARs.

The example demonstrates that IFNA gene family SARs map to the samelocations in U373 glioma cells as those found in BV173 hematopoeticcells. The same pattern of SARs in two different cell types suggest animportance to conserve the chromatin structure of the family. Bothhematopoetic and glial cells are derived from the same precursorpleuripotent stem cells during development. Early in development bloodmacrophages migrate to brain tissue, where they differentiate into glialcells.

The model for the higher order structure of the IFN gene family proposesa series of loop domains (≦10 kb) flanked by SARs. Some small loopscontain an IFN gene with its promoter and regulatory sequences flankedby SARs. Other small loops contain pseudogenes also flanked by SARsthat, at least in some cases, showed weaker scaffold binding. Sincemost, if not all, 22 members of the IFN gene family are flanked by SARs,it is apparent that the duplication of IFN sequences that appear to havegenerated this complex locus in evolution has also involved duplicationof flanking scaffold attachment sites.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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1. A method of diagnosing a tumor in a subject, comprising: obtaining asample comprising or potentially comprising a tumor cell from a subject;obtaining an antibody that binds immunologically to SEQ ID NO:2 or anucleic acid segment that hybridizes to a nucleic acid encoding SEQ IDNO:2; admixing the sample with the antibody or the nucleic acid segment;and determining whether immunologic binding occurs between the antibodyand SEQ ID NO:2 in the sample or hybridization occurs between thenucleic acid segment and a nucleic acid segment encoding SEQ ID NO:2 inthe sample; wherein substantial lack of binding or hybridization isindicative of the presence of leukemia, glioma, melanoma, brain cancer,lung cancer, pancreatic cancer, bladder cancer, head and neck cancer,mesothelioma, ovarian cancer, sarcoma, childhood cancers, or breastcancer in the subject.
 2. The method of claim 1, wherein the samplecomprises one or more cells comprising SEQ ID NO:2 and/or a nucleic acidsegment encoding SEQ ID NO:2.
 3. The method of claim 1, wherein SEQ IDNO:2 and/or nucleic acid segment encoding SEQ ID NO:2 are separated fromthe one or more cells prior to admixing with the antibody or the nucleicacid segment that hybridizes to a nucleic acid segment encoding SEQ IDNO:2.
 4. The method of claim 1, wherein: the antibody that bindsimmunologically to SEQ ID NO:2 or a nucleic acid segment that hybridizesto a nucleic acid segment encoding SEQ ID NO:2 further comprises adetectable label; determining whether immunologic binding occurs betweenthe antibody and SEQ ID NO:2 in the sample or hybridization occursbetween the nucleic acid segment and a nucleic acid segment encoding SEQID NO:2 in the sample comprises detecting the detectable label.
 5. Themethod of claim 4, wherein detectable label is a radio-, enzymatic orfluorescent label.
 6. The method of claim 1, wherein the sample isadmixed with the antibody under conditions effective to allow theformation of immune complexes; and wherein determining whetherimmunologic binding occurs between the antibody and SEQ ID NO:2 in thesample comprises detecting the immune complexes so formed.